Use of heregulin as a growth factor

ABSTRACT

Ligands which bind to the HER2, HER3 and/or HER4 receptors are useful as normal epithelial cell growth factors.

[0001] This is a non-provisional application claiming priority toprovisional application Ser. No. 60/073,866 filed Feb. 4, 1998, theentire disclosure of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to the use of HER2, HER3 and/or HER4ligands, in particular heregulin polypeptides, as epithelial cell growthfactors.

[0004] 2. Description of Background and Related Art

[0005] The HER (ErbB) family belongs to the subclass I receptor tyrosinekinase superfamily and consists of three distinct receptors, HER2, HER3,and HER4. A ligand for this ErbB family is the protein heregulin (HRG),a multidomain containing protein with at least 15 distinct isoforms.

[0006] Transduction of signals that regulate cell growth anddifferentiation is regulated in part by phosphorylation of variouscellular proteins. Protein tyrosine kinases are enzymes that catalyzethis process. Receptor protein tyrosine kinases are believed to directcellular growth via ligand-stimulated tyrosine phosphorylation ofintracellular substrates. Growth factor receptor protein tyrosinekinases of the class I subfamily include the 170 kDa epidermal growthfactor receptor (EGFR) encoded by the erbB1 gene. erbB1 has beencausally implicated in human malignancy. In particular, increasedexpression of this gene has been observed in more aggressive carcinomasof the breast, bladder, lung and stomach.

[0007] The second member of the class I subfamily, p185^(neu), wasoriginally identified as the product of the transforming gene fromneuroblastomas of chemically treated rats. The neu gene (also callederbB2 and HER2) encodes a 185 kDa receptor protein tyrosine kinase.Amplification and/or overexpression of the human HER2 gene correlateswith a poor prognosis in breast and ovarian cancers (Slamon et al.,Science 235:177-182 (1987); and Slamon et al., Science 244:707-712(1989)). Overexpression of HER2 has been correlated with othercarcinomas including carcinomas of the stomach, endometrium, salivarygland, lung, kidney, colon and bladder. Accordingly, Slamon et al. inU.S. Pat No. 4,968,603 describe and claim various diagnostic assays fordetermining HER2 gene amplification or expression in tumor cells. Slamonet al. discovered that the presence of multiple gene copies of HER2oncogene in tumor cells indicates that the disease is more likely tospread beyond the primary tumor site, and that the disease may thereforerequire more aggressive treatment than might otherwise be indicated byother diagnostic factors. Slamon et al. conclude that the HER2 geneamplification test, together with the determination of lymph nodestatus, provides greatly improved prognostic utility.

[0008] A further related gene, called erbB3 or HER3, has also beendescribed. See U.S. Pat. No. 5,183,884; Kraus et al., Proc. Natl. Acad.Sci. USA 86:9193-9197 (1989); EP Pat Appln No 444,961A1; and Kraus etal., Proc. Natl. Acad. Sci. USA 90:2900-2904 (1993). Kraus et al. (1989)discovered that markedly elevated levels of erbB3 mRNA were present incertain human mammary tumor cell lines indicating that erbB3, like erbB1and erbB2, may play a role in human malignancies. Also, Kraus et al.(1993) showed that EGF-dependent activation of the ErbB3 catalyticdomain of a chimeric EGFR/ErbB3 receptor resulted in a proliferativeresponse in transfected NIH-3T3 cells. This is now believed to be theresult of endogenous ErbB1 or ErbB2 in NIH-3T3. Furthermore, theseresearchers demonstrated that some human mammary tumor cell linesdisplay a significant elevation of steady-state ErbB3 tyrosinephosphorylation further indicating that this receptor may play a role inhuman malignancies. The role of erbB3 in cancer has been explored byothers. It has been found to be overexpressed in breast (Lemoine et al.,Br. J. Cancer 66:1116-1121 (1992)), gastrointestinal (Poller et at., J.Pathol. 168:275-280 (1992), Rajkumer et al., J. Pathol. 170:271-278(1993), and Sanidas et al., Int. J. Cancer 54:935-940 (1993)), andpancreatic cancers (Lemoine et al., J. Pathol. 168:269-273 (1992), andFriess et al., Clinical Cancer Research 1:1413-1420 (1995)).

[0009] The class I subfamily of growth factor receptor protein tyrosinekinases has been further extended to include the HER4/Erb4 receptor. SeeEP Pat Appln No 599,274; Plowman et al., Proc. Nalt. Acad. Sci. USA90:1746-1750 (1993); and Plowman et al., Nature 366:473475 (1993).Plowman et al. found that increased HER4 expression closely correlatedwith certain carcinomas of epithelial origin, including breastadenocarcinomas. Diagnostic methods for detection of human neoplasticconditions (especially breast cancers) which evaluate HER4 expressionare described in EP Pat Appln No. 599,274.

[0010] The quest for the activator of the HER2 oncogene has lead to thediscovery of a family of heregulin polypeptides. These proteins appearto result from alternate splicing of a single gene which was mapped tothe short arm of human chromosome 8 by Orr-Urtreger et al., Proc. Natl.Acad. Sci. USA 90:1867-1871(1993). See also Lee and Wood, Genomics,16:790-791 (1993).

[0011] Holmes et al. isolated and cloned a family of polypeptideactivators for the HER2 receptor which they called heregulin-α (HRG-α),heregulin-β1 (HRG-β1), heregulin-β2 (HRG-β2), heregulin-β2-like(HRG-β2-like), and heregulin-β3 (HRG-β3). See Holmes et al., Science256:1205-1210 (1992); WO 92/20798; and U.S. Pat. No. 5,367,060. The 45kDa polypeptide, HRG-α, was purified from the conditioned medium of theMDA-MB-231 human breast cancer cell line. These researchers demonstratedthe ability of the purified heregulin polypeptides to activate tyrosinephosphorylation of the HER2 receptor in MCF7 breast tumor cells.Furthermore, the mitogenic activity of the heregulin polypeptides onSK-BR-3 cells (which express high levels of the HER2 receptor) wasillustrated. Like other growth factors which belong to the EGF family,soluble HRG polypeptides appear to be derived from a membrane boundprecursor (called pro-HRG) which is proteolytically processed to releasethe 45kDa soluble form. These pro-HRGs lack a N-terminal signal peptide.

[0012] While heregulins are substantially identical in the first 213amino acid residues, they are classified into two major types, α and β,based on two variant EGF-like domains which differ in their C-terminalportions. Nevertheless, these EGF-like domains are identical in thespacing of six cysteine residues contained therein. Based on an aminoacid sequence comparison, Holmes et al. found that between the first andsixth cysteines in the EGF-like domain, HRGs were 45% similar toheparin-binding EGF-like growth factor (HB-EGF), 35% identical toamphiregulin (AR), 32% identical to TGF-α, and 27% identical to EGF.

[0013] The 44 kDa neu differentiation factor (NDF), which is the ratequivalent of human HRG, was first described by Peles et al., Cell,69:205-216 (1992); and Wen et al., Cell, 69:559-572 (1992). Like the HRGpolypeptides, NDF has an immunoglobulin (Ig) homology domain followed byan EGF-like domain and lacks a N-terminal signal peptide. Subsequently,Wen et al., Mol. Cell. Biol., 14(3):1909-1919 (1994) carried out“exhaustive cloning” to extend the family of NDFs. This work revealedsix distinct fibroblastic pro-NDFs. Adopting the nomenclature of Holmeset al., the NDFs are classified as either α or β polypeptides based onthe sequences of the EGF-like domains. Isoforms 1 to 4 are characterizedon the basis of the variable justamembrane stretch (between the EGF-likedomain and transmembrane domain). Also, isoforms a, b and c aredescribed which have variable length cytoplasmic domains. Theseresearchers conclude that different NDF isoforms are generated byalternative splicing and perform distinct tissue-specific functions. Seealso EP 505 148; WO 93/22424; and WO 94128133 concerning NDF.

[0014] Falls et al., Cell, 72:801-815 (1993) describe another member ofthe heregulin family which they call acetylcholine receptor inducingactivity (ARIA) polypeptide. The chicken-derived ARIA polypeptidestimulates synthesis of muscle acetylcholine receptors. See also WO94/08007. ARIA is a β-type heregulin and lacks the entire spacer regionrich in glycosylation sites between the Ig-like domain and EGF-likedomain of HRGα, and HRGβ1-β3.

[0015] Marchionni et al., Nature, 362:312-318 (1993) identified severalbovine-derived proteins which they call glial growth factors (GGFs).These GGFs share the Ig-like domain and EGF-like domain with the otherheregulin proteins described above, but also have an amino-terminalkringle domain. GGFs generally do not have the complete glycosylatedspacer region between the Ig-like domain and EGF-like domain. Only oneof the GGFs, GGFII, possessed a N-terminal signal peptide. See also WO92/18627; WO 94/00140; WO 94/04560; WO 94/26298; and WO 95/32724 whichrefer to GGFs and uses thereof.

[0016] Ho et al. in J. Biol. Chem. 270(4):14523-14532 (1995) describeanother member of the heregulin family called sensory and motorneuron-derived factor (SMDF). This protein has an EGF-like domaincharacteristic of all other heregulin polypeptides but a distinctN-terminal domain. The major structural difference between SMDF and theother heregulin polypeptides is the lack in SMDF of the Ig-like domainand the “glyco” spacer characteristic of all the other heregulinpolypeptides. Another feature of SMDF is the presence of two stretchesof hydrophobic amino acids near the N-terminus.

[0017] While the heregulin polypeptides were first identified based ontheir ability to activate the HER2 receptor (see Holmes et al., supra),it was discovered that certain ovarian cells expressing neu andneu-transfected fibroblasts did not bind or crosslink to NDF, nor didthey respond to NDF to undergo tyrosine phosphorylation (Peles et al.,EMBO J. 12:961-971 (1993)). This indicated another cellular componentwas necessary for conferring full heregulin responsiveness. Carraway etal. subsequently demonstrated that ¹²⁵I-rHRGβ1₁₇₇₋₂₄₄ bound to NIH-3T3fibroblasts stably transfected with bovine erbB3 but not tonon-transfected parental cells. Accordingly, they conclude that ErbB3 isa receptor for HRG and mediates phosphorylation of intrinsic tyrosineresidues as well as phosphorylation of ErbB2 receptor in cells whichexpress both receptors. Carraway et al., J. Biol. Chem.269(19):14303-14306 (1994). Sliwkowski et al., J. Biol. Chem.269(20):14661-14665 (1994) found that cells transfected with HER3 aloneshow low affinities for heregulin, whereas cells transfected with bothHER2 and HER3 show higher affinities.

[0018] This observation correlates with the “receptor cross-talking”described previously by Kokai et al, Cell 58:287-292 (1989); Stern etal., EMBO J. 7:995-1001 (1988); and King et al., 4:13-18 (1989). Theseresearchers found that binding of EGF to the EGFR resulted in activationof the EGFR kinase domain and cross-phosphorylation of p₁₈₅ ^(HER2).This is believed to be a result of ligand-induced receptorheterodimerization and the concomitant cross-phosphorylation of thereceptors within the heterodimer (Wada et al., Cell 61:1339-1347(1990)).

[0019] Plowman and his colleagues have similarly studiedp185^(HER4)/p185^(HEP2) activation. They expressed p185^(HER2) alone,p185^(HER4) alone, or the two receptors together in human T lymphocytesand demonstrated that heregulin is capable of stimulating tyrosinephosphorylation of p185^(HER4), but could only stimulate p185^(HER2)phosphorylation in cells expressing both receptors. Plowman et al.,Nature 336:473475 (1993).

[0020] The biological role of heregulin has been investigated by severalgroups. For example, Falls et al., (discussed above) found that ARIAplays a role in myotube differentiation, namely affecting the synthesisand concentration of neurotransmitter receptors in the postsynapticmuscle cells of motor neurons. Corfas and Fischbach demonstrated thatARIA also increases the number of sodium channels in chick muscle.Corfas and Fischbach, J. Neuroscience, 13(5): 2118-2125 (1993). It hasalso been shown that GGFII is mitogenic for subconfluent quiescent humanmyoblasts and that differentiation of clonal human myoblasts in thecontinuous presence of GGFII results in greater numbers of myotubesafter six days of differentiation (Sklar et al., J. Cell Biochem., Abst.W462, 18D, 540 (1994)). See also WO 94/26298 published Nov. 24, 1994.

[0021] Holmes et al., supra, found that HRG exerted a mitogenic effecton mammary cell lines (such as SK-BR-3 and MCF-7). The mitogenicactivity of GGFs on Schwann cells has also been reported. See, e.g.,Brockes et al., J. Biol. Chem. 255(18):8374-8377 (1980); Lemke andBrockes, J. Neurosci. 4:75-83 (1984 Lemke and Brockes, J. Neurosci.4:75-83 (1984); Brockes et al., Ann. Neurol. 20(3):317-322 (1986);Brockes, J., Methods in Enzym., 147: 217-225 (1987) and Marchionni etal., supra. Schwann cells constitute important glial cells which providemyelin sheathing around the axons of neurons, thereby forming individualnerve fibers. Thus, it is apparent that Schwann cells play an importantrole in the development, function and regeneration of peripheral nerves.The implications of this from a therapeutic standpoint have beenaddressed by Levi et a!., J. Neuroscience 14(3):1309-1319 (1994). Leviet al. discuss the potential for construction of a cellular prosthesiscomprising human Schwann cells which could be transplanted into areas ofdamaged spinal cord. Methods for culturing Schwann cells ex vivo havebeen described. See WO 94/00140 and Li et al., J. Neuroscience16(6):2012-2019 (1996).

[0022] Pinkas-Kramarski et aL found that NDF seems to be expressed inneurons and glial cells in embryonic and adult rat brain and primarycultures of rat brain cells, and suggested that it may act as a survivaland maturation factor for astrocytes (Pinkas-Kramarski et a., PNAS, USA91:9387-9391 (1994)). Meyer and Birchmeier, PNAS, USA 91:1064-1068(1994) analyzed expression of heregulin during mouse embryogenesis andin the perinatal animal using in situ hybridization and RNase protectionexperiments. See also Meyer et al., Development 124(18):3575-3586(1997). These authors conclude that, based on expression of thismolecule, heregulin plays a role in vivo as a mesenchymal and neuronalfactor. Similarly, Danilenko et al., Abstract 3101, FASEB 8(4-5):A535(1994); Danilenko et al., Journal of Clinical Investigation 95(2):842-851 (1995), found that the interaction of NDF and the HER2 receptoris important in directing epidermal migration and differentiation duringwound repair.

[0023] Ram et al., Journal of Cellular Physiology 163:589-596 (1995)evaluated the mitogenic activity of NDF on the immortalized humanmammary epithelial cell line MCF-10A. Danilenko et al., J. Clin. Invest.95:842-851 (1995) investigated whether NDF would influence epidermalmigration in an in vivo model of excisional deep partial-thickness woundrepair. It is reported that there were no statistically significantdifferences in proliferating basal and superbasal keratinocytes incontrol wounds vs. wounds treated with rhNDF-α₂. Marikovsky et al.,Oncogene 10:1403-1411 (1995), studied the proliferative responses of ananeuploid BALB/MK continuous keratinocyte cell line and evaluated theeffects of α- and β-isoforms of NDF on epidermal keratinocytes.

[0024] The relationship between the structure and function of newproteins can be investigated using any of a variety of availablemutational analysis techniques. Examples of such techniques includealanine scanning mutagenesis and phagemid display. Alanine scanning canbe used to identify active residues (i.e., residues that have asignificant effect on protein function) in a protein or protein domain.For example, Cunningham and Wells used alanine scanning to identifyresidues in human growth hormone that were important for binding itsreceptor. Cunningham and Wells, Science 244:1081-1085 (1989). In alaninescanning, a gene encoding the protein or domain to be scanned isinserted into an expression vector, and mutagenesis is carried out togenerate a series of vectors that encode proteins or domains in whichsequential residues are converted to alanine. The encoded proteins ordomain are expressed from these vectors, and the activities of thealanine-substituted variants are then tested to identify those withaltered activity. An alteration in activity indicates that the residueat the alanine-substituted position is an active residue.

[0025] Phagemid display was developed to allow the screening of a largenumber of variant polypeptides for a particular binding activity. Smithand Parmley demonstrated that foreign peptides can be “displayed”efficiently on the surface of filamentous phage by inserting short genefragments into gene III of the fd phage. Smith, Science 228:1315-1317(1985); Parmley and Smith, Gene 73:305-318 (1985). The gene III coatprotein is present in about five copies at one end of the phageparticle. The modified phage were termed “fusion phage” because theydisplayed the foreign peptides fused to the gene III coat protein. Aseach fusion phage particle displayed approximately five copies of thefusion protein, this mode of phage display was termed “polyvalentdisplay.”

[0026] Scott et al. and Cwirla et al. showed that fusion phage librariescould be screened by sequential affinity selections known as “panning.”Scott et al., Science 249:386-390 (1990); Cwirla et al., PNAS USA87:6378-6382 (1990). However, early efforts to select high affinityfusion phage failed, presumably due to the polyvalence of the phageparticles. This problem was solved with the development of a“monovalent” phage display system in which the fusion protein isexpressed at a low level from a phagemid and a helper phage provides alarge excess of wild-type coat protein. Bass et al., Proteins 8:309-314(1990); Lowman et at., Biochem. 30:10832-10838 (1991). Monovalent phagedisplay can be used to generate and screen a large number of variantpolypeptides to isolate those that bind with high affinity to a targetof interest.

[0027] Approximately 50,000 infants are born in the United States everyyear with birth weights, less than 1.5 kg. About two thirds of thesevery low birth weight infants have evidence of pulmonary immaturitymanifested as respiratory distress shortly after birth. The majority ofthese infants require mechanical ventilation. Respiratory distresssyndrome, caused by insufficient pulmonary surfactant production, aswell as structural immaturity of the lung, is responsible forrespiratory difficulties observed in these prematurely born neonates.Well developed alveoli are necessary to provide efficient oxygentransfer from the air-liquid interface of the lung to the systemiccirculation. Surfactant proteins are critical in reducing the alveolarsurface tension at low lung volumes and preventing alveolar collapse.

[0028] A need continues to exist for a method of treatment forrespiratory distress syndrome and other diseases associated withimmature lung development and low lung surfactant production.

SUMMARY OF THE INVENTION

[0029] In general an object of the invention is to provide a method ofinducing epithelial cell growth and development for the purpose ofpromoting repair and healing of tissue damage or injury.

[0030] Accordingly, one object of this invention is to provide a methodof treating respiratory distress syndrome in patients, primarily humanpatients, in need of such treatment. A further object is to provide amethod of inducing lung epithelial cell growth and development. Afurther object is to provide a method of increasing lung surfactantprotein A production in the lung of persons with impaired oxygentransfer in the lung alveoli. This invention is useful in treatinginfants/neonates with respiratory distress as well as youth and adultswith poor lung function due to lung injury or damage.

[0031] In one aspect of this invention, it has now been discovered thatthese objects and the broader objective of treating conditionsassociated with epithelial cell damage and injury are achieved byadministering to a patient in need of such treatment an effective amountof a heregulin ligand, preferably a polypeptide or fragment thereof.These heregulin (HRG) polypeptides, include HRG-α, HRG-β1, HRG-β2,HRG-β3 and other HRG polypeptides which cross-react with antibodiesdirected against these family members and/or which are substantiallyhomologous as defined below and includes HRG variants such as N-terminaland C-terminal fragments thereof. A preferred HRG is the liganddisclosed in FIGS. 1A-1D and further designated HRG-α. Other preferredHRGs are the ligands disclosed in FIGS. 2A-2E, and designated HRG-β1;disclosed in FIGS. 3A-3E designated HRG-β2; and disclosed in FIGS. 4A-4Cdesignated HRG-β3.

[0032] In another aspect, the invention provides a method in which HRGagonist antibodies are administered to achieve the objects of theinvention. In this embodiment, HER2/HER3 or fragments thereof (whichalso may be synthesized by in vitro methods) are fused (by recombinantexpression or an in vitro peptidyl bond) to an immunogenic polypeptideand this fusion polypeptide, in turn, is used to raise antibodiesagainst a HER2/HER3 epitope. Agonist antibodies are recovered from theserum of immunized animals. Alternatively, monoclonal antibodies areprepared from in vitro cells or in vivo immunized animals inconventional fashion. If desired, the agonist antibodies may be obtainedby phage display selection from a phage library of antibodies orantibody fragments. Preferred antibodies identified by routine screeningwill bind to the receptor, but will not substantially cross-react withany other known ligands such as EGF, and will activate the HER receptorsHER2, HER3 and/or HER4. In addition, antibodies may be selected that arecapable of binding specifically to individual family members of HRGfamily, e.g. HRG-α, HRG-β1, HRG-β2, HRG-β3, and which are agoniststhereof.

[0033] In general, the invention is a method of regenerating and/orrepairing epithelial cell injury by stimulating growth and proliferationof epithelial cells, in particular ductal and ciliated epithelial cells.The epithelial cells may be injured by many types of insults, forexample, injury due to surgical incision or resection, chemical or smokeinhalation or aspiration, chemical or biochemical ulceration, celldamage due to viral or bacterial infection, etc. The epithelial cellswhich may be affected by the method of the invention include anyepithelial cell which expresses HER2, HER3 and/or HER4; suitable cellsare located, for example, in the lung, gastric mucosa, endometrium,oviducts, mammary glands, pancreas, salivary glands, etc. The method ofthe invention stimulates growth and proliferation of the epithelialcells, repairing and re-establishing the cellular barriers of organs andallowing the affected tissues to develop normal physiological functionsmore quickly. For example, lung epithelial cells are damaged byinhalation of smoke resulting in emphysema. Treatment of the lung cellsby the method of the invention regenerates the barrier layer of lungepithelial cells, improves oxygenation and speeds the development of abarrier to infection. Similarly, cell damage due to aspiration ofgastric acid can be treated by the method of the invention to facilitateregeneration of epithelial cells.

[0034] Accordingly, one embodiment of the invention is a method ofinducing lung epithelial cell growth and development by contacting alung epithelial cell which expresses HER2, HER3 and/or HER4 receptorswith an effective amount of a HER2, HER3 and/or HER4 activating ligand.

[0035] Another embodiment is a method of increasing lung surfactantprotein A in a patient by administering to a patient in need thereof aneffective amount of a HER2, HER3 and/or HER4 activating ligand.

[0036] A further embodiment is a method of treating respiratory distressby administering to a patient in need thereof an effective amount of aHER2, HER3 and/or HER4 activating ligand.

[0037] A further embodiment is a method of treating emphysema byadministering to a patient in need thereof an effective amount of aHER2, HER3 and/or HER4 activating ligand.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIGS. 1A-1D show the deduced amino acid sequence (SEQ ID NO:1)for the cDNA sequence (SEQ ID NO:2) contained in a clone obtainedaccording to U.S. Pat. No. 5,367,060. The initiating methionine (Met) ofHRG-α is at position 45.

[0039] FIGS. 2A-2E show the deduced amino acid sequence (SEQ ID NO:3)and cDNA sequence (SEQ ID NO:4) of a potential coding sequence of aclone obtained according to U.S. Pat. No. 5,367,060 for HRG-β1. Theinitiating Met is at M31.

[0040] FIGS. 3A-3E show the deduced amino acid sequence (SEQ ID NO:5)and cDNA sequence (SEQ ID NO:6) of a nucleotide sequence of a cloneobtained according to U.S. Pat. No. 5,367,060 for HRG-β2.

[0041] FIGS. 4A-4C show the deduced amino acid sequence (SEQ ID NO:7)and cDNA sequence (SEQ ID NO:8) of a nucleotide sequence of a cloneobtained according to U.S. Pat. No. 5,367,060 for HRG-β3.

[0042] FIGS. 5A-5D show the deduced amino acid sequence (SEQ ID NO:9)and cDNA sequence (SEQ ID NO:10) of a nucleotide sequence of a cloneobtained according to U.S. Pat. No. 5,367,060 for HRG-β2-like protein.

[0043] FIGS. 6A-6C show a comparison of the amino acid homologies ofseveral known heregulins α, β1, β2, β2-like and β3 in descending orderand illustrates the amino acid insertions, deletions, and substitutionsthat characterize these forms of HRG (SEQ ID NOS: 1, 3, 5, 9, and 7).

[0044] FIGS. 7A-7C show the deduced amino acid sequence (SEQ ID NO:11)and cDNA sequence (SEQ ID NO:12) of γ-HRG obtained as described in U.S.Ser. No. 08/891,845. The hydrophobic region is underlined. The EGF-likedomain is shaded, cysteine residues in the EGF-like domain are circled.N-linked glycosylation sites are marked above the nucleic acid sequencewith a (•).

[0045]FIG. 8 shows the cDNA sequence (SEQ ID NO:13) and amino acidsequence (SEQ ID NO:14) of SMDF obtained as described in U.S. Ser. No.08/339,517. An EGF-like domain and the apolar and uncharged domains(i.e. “apolar I” consisting of residues from about 48-62 and “apolar 11”consisting of residues from about 76-100) are underlined. Cysteines inthe EGF-like domain and in the “cysteine knot” in the unique N-terminaldomain (“NTD-cys knot”) are boxed. The stop codon is denoted by theletter “O”.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] HRG ligands, in particular polypeptides and agonist antibodiesthereof, have affinity for and stimulate the HER2, HER3 and/or HER4receptors or combinations thereof in autophosphorylation. Includedwithin the definition of HRG ligands, in addition to HRG-α, HRG-β1,HRG-β2, HRG-β3 and HRG-β2-like, are other polypeptides binding to theHER2, HER3 and/or HER4 receptor, which bear substantial amino acidsequence homology to HRG-α or HRG-β1. Such additional polypeptides fallwithin the definition of HRG as a family of polypeptide ligands thatbind to the HER2, HER3 and/or HER4 receptors.

[0047] Heregulin polypeptides bind with varying affinities to the HER2,HER3 and/or HER4 receptors. Generally, the HER3 and HER4 receptors arebound with high affinity. It is also known that heterodimerization ofHER2 with HER3 and of HER2 with HER4 occurs with subsequent receptorcross-phosphorylation as described above. In the present invention,epithelial cell growth and/or proliferation is induced when a heregulinprotein interacts and binds with an individual receptor molecule or areceptor dimer such that receptor phosphorylation is induced. Bindingand activation of HER2, HER3, HER4 or combinations thereof, therefore,is meant to include activation of any form of the receptor necessary forreceptor activation and biologic function including monomeric receptorand dimeric receptor forms. Dimeric receptor forms may be referred tobelow, for example, as HER2/HER3, HER2/HER4, and HER3/HER4.

[0048] I. Definitions

[0049] In general, the following words or phrases have the indicateddefinition when used in the description, examples, and claims.

[0050] “Heregulin” (HRG) ligand is defined herein to be any isolatedligand, preferably a polypeptide sequence which possesses a biologicalproperty of a naturally occurring HRG polypeptide. Ligands within thescope of this invention include the NDF, ARIA and GGF growth factorheregulin proteins identified above as well as the SMDF and HRGpolypeptides discussed in detail herein. These isolated NDF, ARIA andGGF heregulin polypeptides are well known in the art. HRG includes thepolypeptides shown in FIGS. 1A-1D, 2A-2E, 3A-3E, 4A-4C, 5A-5D, 6A-6C,7A-7C and 8 and mammalian analogues thereof. Included are HRG variantssuch as the γ-HRG described in U.S. application Ser. No. 08/891,845filed Jul. 10, 1997, the variants described in Ser. No. 08/799,054 filedFeb. 10, 1997 and the SMDF variants described in Ser. No. 08/339,517filed Nov. 14, 1994 (WO 96/15244). These applications are incorporatedherein in their entirety. These variants can be prepared by the methodsdescribed below, optionally together with alanine scanning and phagedisplay techniques known in the art. Cunningham and Wells, Science244:1081-85 (1989); Bass et al., Proteins 8:309-14 (1990); Lowman etal., Biochem. 30:10832-38 (1991).

[0051] The term a “normal” epithelial cell means an epithelial cellwhich is not transformed, i.e., is non-cancerous and/ornon-immortalized. Further, the normal epithelial cell is preferably notaneuploid. Aneuploidy exists when the nucleus of a cell does not containan exact multiple of the haploid number of chromosomes, one or morechromosomes being present in greater or lesser number than the rest.Typical properties of transformed cells which fall outside the scope ofthis invention include the ability to form tumors when implanted intoimmune-deprived mice (nude mice), the go -ability to grow in suspensionor in semi-solid media such as agar, a loss of contact inhibitionallowing piling up of cells into colonies or foci, a loss of dependenceon growth factors or serum, cell death if cells are inhibited fromgrowing, and disorganization of actin filaments. Specifically includedwithin the invention are normal epithelial cells which will not formtumors in mice, grow attached to plastic or glass (are anchoragedependent), exhibit contact inhibition, require serum-containinghormones and growth factors, remain viable if growth is arrested by lackof serum, and contain well-organized actin filaments. Although thenormal epithelial cells are preferably not cultured cells, also suitablefor the invention are non-transformed, non-immortalized epithelial cellsisolated from mammalian tissue. These isolated cells may be cultured forseveral generations (up to about 10 or even 50 generations) in thepresence of a heregulin in order to induce growth and/or proliferationof the isolated epithelial cell sample, that is, to expand the sample.The expanded sample can then be reintroduced into the mammal for thepurpose of repopulating the epithelial cell tissue(re-epithelialization). This is particularly useful for repairing tissueinjury or damage.

[0052] An “epithelial” cell is a cell located in a cellular, avascularlayer covering the free surface (cutaneous, mucous or serous) of anorgan or lining a tube or cavity of an animal body. Lung epithelialcells include bronchial epithelial cells, Type II cells and Clara cells.The term “epithelial cell” as used herein is consistent with the artrecognized definition of epithelial cells in epithelium. See, forexample, the definition in Taber's Encyclopedic Medical Dictionary,Edition 12, (1973) F.A. Davis Company, publisher.

[0053] “Biological property” for the purposes herein means an in vivobiologic or antigenic function or activity that is directly orindirectly performed by an HRG sequence (whether in its native ordenatured conformation), or by any subsequence thereof. Biologicfunctions include receptor binding, any enzyme activity or enzymemodulatory activity, any carrier binding activity, any hormonalactivity, any activity in promoting or inhibiting adhesion of cells toextracellular matrix or cell surface molecules, or any structural role.However, biologic functions do not include antigenic functions, i.e.possession of an epitope or antigenic site that is capable ofcross-reacting with antibodies raised against a naturally occurring HRGpolypeptide.

[0054] “Biologically active” HRG is defined herein as a polypeptidesharing a biologic function of an HRG sequence which may (but need not)in addition possess an antigenic function. A principal known effect orfunction of HRG is as a ligand polypeptide having a qualitativebiological activity of binding to HER2, HER3 and/or HER4 resulting inthe activation of the receptor tyrosine kinase (an “activating ligand”).One test for activating ligands is the HRG tyrosine autophosphorylationassay described below. Included within the scope of HRG as that term isused herein are HRG having translated mature amino acid sequences of thecomplete human HRG as set forth herein; deglycosylated or unglycosylatedderivatives of HRG, amino acid sequence variants of HRG sequence, andderivatives of HRG, which are capable of exhibiting a biologicalproperty in common with HRG. While native HRG is a membrane-boundpolypeptide, soluble forms, such as those forms lacking a functionaltransmembrane domain, are also included within this definition. Inparticular, included are polypeptide fragments of HRG sequence whichhave an N-terminus at any residue from about S216 to about A227, and itsC-terminus at any residue from about K268 to about R286, and thehomologous sequences shown in FIGS. 6A-C, hereinafter referred tocollectively for all HRGs as the growth factor domain (GFD).

[0055] “Antigenically active” HRG is defined as a polypeptide thatpossesses an antigenic function of an HRG and which may (but need not)in addition possess a biologic function.

[0056] In preferred embodiments, antigenically active HRG is apolypeptide that binds with an affinity of at least about 10⁻⁹ l/mole toan antibody raised against a naturally occurring HRG sequence.Ordinarily the polypeptide binds with an affinity of at least about 10⁻⁸l/mole. Most preferably, the antigenically active HRG is a polypeptidethat binds to an antibody raised against one of HRGs in its nativeconformation. HRG in its native conformation generally is HRG as foundin nature which has not been denatured by chaotropic agents, heat orother treatment that substantially modifies the three dimensionalstructure of HRG as determined, for example, by migration onnonreducing, nondenaturing sizing gels. Antibody used in thisdetermination may be rabbit polyclonal antibody raised by formulatingnative HRG from a non-rabbit species in Freund's complete adjuvant,subcutaneously injecting the formulation, and boosting the immune aresponse by intraperitoneal injection of the formulation until the titerof anti-HRG antibody plateaus.

[0057] Ordinarily, biologically or antigenically active HRG will have anamino acid sequence having at least 75% amino acid sequence identitywith a given HRG sequence, more preferably at least 80%, even morepreferably at least 90%, and most preferably at least 95%. Identity orhomology with respect to an HRG sequence is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical with HRG residues in the HRG of FIGS. 6A-6C, after aligningthe sequences and introducing gaps, if necessary, to achieve the maximumpercent homology, and not considering any conservative substitutions aspart of the sequence identity. None of N-terminal, C-terminal orinternal extensions, deletions, or insertions into HRG sequence shall beconstrued as affecting homology.

[0058] Thus, the biologically active and antigenically active HRGpolypeptides that are the subject of this invention include each entireHRG sequence; fragments thereof having a consecutive sequence of atleast 5, 10, 15, 20, 25, 30 or 40 amino acid residues from HRG sequence;amino acid sequence variants of HRG sequence wherein an amino acidresidue has been inserted N- or C-terminal to, or within, HRG sequenceor its fragment as defined above; amino acid sequence variants of HRGsequence or its fragment as defined above has been substituted byanother residue. HRG polypeptides include those containing predeterminedmutations by, e.g., site-directed or PCR mutagenesis, and other animalspecies of HRG polypeptides such as rabbit, rat, porcine, non-humanprimate, equine, murine, and ovine HRG and alleles or other naturallyoccurring variants of the foregoing and human sequences; derivatives ofHRG or its fragments as defined above wherein HRG or its fragments havebeen covalently modified by substitution, chemical, enzymatic, or otherappropriate means with a moiety other than a naturally occurring aminoacid (for example a detectable moiety such as an enzyme orradioisotope); glycosylation variants of HRG (insertion of aglycosylation site or deletion of any glycosylation site by deletion,insertion or substitution of appropriate amino acid); and soluble formsof HRG, such as HRG-GFD or those that lack a functional transmembranedomain.

[0059] “Isolated” means a ligand, such as HRG, which has been identifiedand separated and/or recovered from a component of its naturalenvironment. Contaminant components of its natural environment arematerials which would interfere with diagnostic or therapeutic uses forHRG, and may include proteins, hormones, and other substances. Inpreferred embodiments, HRG will be purified (1) to greater than 95% byweight of protein as determined by the Lowry method or other validatedprotein determination method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of the bestcommercially available amino acid sequenator marketed on the filing datehereof, or (3) to homogeneity by SDS-PAGE using Coomassie blue or,preferably, silver stain. Isolated HRG includes HRG in situ withinrecombinant cells since at least one component of HRG naturalenvironment will not be present. Isolated HRG includes HRG from onespecies in a recombinant cell culture of another species since HRG insuch circumstances will be devoid of source polypeptides. Ordinarily,however, isolated HRG will be prepared by at least one purificationstep.

[0060] In accordance with this invention, HRG nucleic acid is RNA or DNAcontaining greater than ten bases that encodes a biologically orantigenically active HRG, is complementary to nucleic acid sequenceencoding such HRG, or hybridizes to nucleic acid sequence encoding suchHRG and remains stably bound to it under stringent conditions.

[0061] Preferably, HRG nucleic acid encodes a polypeptide sharing atleast 75% sequence identity, more preferably at least 80%, still morepreferably at least 85%, even more preferably at 90%, and mostpreferably 95%, with an HRG sequence. Preferably, the HRG nucleic acidthat hybridizes contains at least 20, more preferably at least about 40,and most preferably at least about 90 bases.

[0062] Isolated HRG nucleic acid includes a nucleic acid that isidentified and separated from at least one containment nucleic acid withwhich it is ordinarily associated in the natural source of HRG nucleicacid. Isolated HRG nucleic acid thus is present in other than in theform or setting in which it is found in nature. However, isolated HRGencoding nucleic acid includes HRG nucleic acid in ordinarilyHRG-expressing cells where the nucleic acid is in a chromosomal locationdifferent from that of natural cells or is otherwise flanked by adifferent DNA sequence than that found in nature. Nucleic acid encodingHRG may be used in specific hybridization assays, particularly thoseportions of HRG encoding sequence that do not hybridize with other knownDNA sequences. “Stringent conditions” are those that (1) employ lowionic strength and high temperature for washing, for example, 0.015 MNACl/0.0015 M sodium citrate/0/1% NaDodSO₄ at 50° C.; (2) employ duringhybridization a denaturing agent such as formamide, for example, 50%(vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1%polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750mM NaCl, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide,5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmonsperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., withwashes at 420 C in 0.2×SSC and 0.1% SDS.

[0063] The term “control sequences” refers to DNA sequences necessaryfor the expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, aribosome binding site, and possibly, other as yet poorly understoodsequences. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

[0064] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein that participates inthe secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous and, in the case of a secretory leader, contiguous and inreading phase. However enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, then synthetic oligonucleotide adaptors or linkersare used in accord with conventional practice.

[0065] An “exogenous” element is defined herein to mean nucleic acidsequence that is foreign to the cell, or homologous to the cell but in aposition within the host cell nucleic acid in which the element isordinarily not found.

[0066] As used herein, the expressions “cell”, “cell line”, and “cellculture” are used interchangeably, and all such designations includeprogeny. Thus, the words “transformants” and “transformed cells” includethe primary subject cell and cultures derived therefrom without regardfor the number of transfers. It is also understood that all progeny maynot be precisely identical in DNA content, due to deliberate orinadvertent mutations. Mutant progeny that have the same function orbiological activity as screened for in the originally transformed cellare included. It will be clear from the context where distinctdesignations are intended.

[0067] “Plasmids” are designated by a lower case “p” preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare commercially available, are publicly available on an unrestrictedbasis, or can be constructed from such available plasmids in accord withpublished procedures. In addition, other equivalent plasmids are knownin the art and will be apparent to the ordinary artisan.

[0068] “Restriction enzyme digestion” of DNA refers to catalyticcleavage of the DNA with an enzyme that acts only at certain locationsin the DNA. Such enzymes are called restriction endonucleases, and thesites for which each is specific is called a restriction site. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors, and other requirements asestablished by the enzyme suppliers are used. Restriction enzymescommonly are designated by abbreviations composed of a capital letterfollowed by other letters representing the microorganism from which eachrestriction enzyme originally was obtained, and then a numberdesignating the particular enzyme. In general, about 1 mg of plasmid orDNA fragment is used with about 1-2 units of enzyme in about 20 ml ofbuffer solution. Appropriate buffers and substrate amounts forparticular restriction enzymes are specified by the manufacturer.Incubation of about 1 hour at 37° C. is ordinarily used, but may vary inaccordance with the supplier's instructions. After incubation, proteinor polypeptide is removed by extraction with phenol and chloroform, andthe digested nucleic acid is recovered from the aqueous fraction byprecipitation with ethanol. Digestion with a restriction enzyme may befollowed with bacterial alkaline phosphatase hydrolysis of the terminal5′ phosphates to prevent the two restriction cleaved ends of a DNAfragment from “circularizing” or forming a closed loop that would impedeinsertion of another DNA fragment at the restriction site. Unlessotherwise stated, digestion of plasmids is not followed by 5′ terminaldephosphorylation. Procedures and reagents for dephosphorylation areconventional as described in sections 1.56-1.61 of Sambrook et al.,(Molecular Cloning: A Laboratory Manual New York: Cold Spring HarborLaboratory Press, 1989).

[0069] “Recovery” or “isolation” of a given fragment of DNA from arestriction digest means separation of the digest on polyacrylamide oragarose gel by electrophoresis, identification of the fragment ofinterest by comparison of its mobility versus that of marker DNAfragments of known molecular weight, removal of the gel sectioncontaining the desired fragment, and separation of the gel from DNA.This procedure is known generally. For example, see Lawn et al., NucleicAcids Res. 9:6103-6114 (1981), and Goeddet et al., Nucleic Acids Res.8:4057 1980).

[0070] “Northern analysis” is a method used to identify RNA sequencesthat hybridize to a known probe such as an oligonucleotide, DNAfragment, cDNA or fragment thereof, or RNA fragment. The probe islabeled with a radioisotope such as ³²p, or by biotinylation, or with anenzyme. The RNA to be analyzed is usually electrophoretically separatedon an agarose or polyacrylamide gel, transferred to nitrocellulose,nylon, or other suitable membrane, and hybridized with the probe, usingstandard techniques well known in the art such as those described insections 7.39-7.52 of Sambrook et al., supra.

[0071] “Ligation” refers to the process of forming phosphodiester bondsbetween two nucleic acid fragments. To ligate the DNA fragmentstogether, the ends of the DNA fragments must be compatible with eachother. In some cases, the ends will be directly compatible afterendonuclease digestion. However, it may be necessary to first convertthe staggered ends commonly produced after endonuclease digestion toblunt ends to make them compatible for ligation. To blunt the ends, theDNA is treated in a suitable buffer for at least 15 minutes at 15° C.with about 10 units of the Klenow fragment of DNA polymerase I or T4 DNApolymerase in the presence of the four deoxyribonucleotidetriphosphates. The DNA is then purified by phenol-chloroform extractionand ethanol precipitation. The DNA fragments that are to be ligatedtogether are put in solution in about equimolar amounts. The solutionwill also contain ATP, ligase buffer, and a ligase such as T4 DNA ligaseat about 10 units per 0.5 mg of DNA. If the DNA is to be ligated into avector, the vector is first linearized by digestion with the appropriaterestriction endonuclease(s). The linearized fragment is then treatedwith bacterial alkaline phosphatase, or calf intestinal phosphatase toprevent self-ligation during the ligation step.

[0072] “Preparation” of DNA from cells means isolating the plasmid DNAfrom a culture of the host cells. Commonly used methods for DNApreparation are the large and small-scale plasmid preparations describedin sections 1.25-1.33 of Sambrook et al., supra. After preparation ofthe DNA, it can be purified by methods well known in the art such asthat described in section 1.40 of Sambrook et al., supra.

[0073] “Oligonucleotides” are short-length, single- or double-strandedpolydeoxynucleotides that are chemically synthesized by known methods(such as phosphotriester, phosphite, or phosphoramidite chemistry, usingsolid phase techniques such as described in EP 266,032, published May 4,1988, or via deoxynucleoside H-phosphonate intermediates as described byFroehler et al., Nucl. Acids Res. 14:5399-5407, 1986. They are thenpurified on polyacrylamide gels.

[0074] The technique of “polymerase chain reaction,” or “PCR,” as usedherein generally refers to a procedure wherein minute amounts of aspecific piece of nucleic acid, RNA and/or DNA, are amplified asdescribed in U.S. Pat. No. 4,683,195, issued Jul. 28, 1987. Generally,sequence information from the ends of the region of interest or beyondneeds to be available, such that oligonucleotide primers can bedesigned; these primers will be identical or similar in sequence toopposite strands of the template to be amplified. The 5′ terminalnucleotides of the two primers may coincide with the ends of theamplified material. PCR can be used to amplify specific RNA sequences,specific DNA sequences from total genomic DNA, and cDNA transcribed fromtotal cellular RNA, bacteriophage or plasmid sequences, etc. Seegenerally Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51: 263(1987); Erlich, ed., PCR Technology, (Stockton Press, NY, 1989). As usedherein, PCR is considered to be one, but not the only, example of anucleic acid polymerase reaction method for amplifying a nucleic acidtest sample, comprising the use of a known nucleic acid (DNA or RNA) asa primer, and utilizes a nucleic acid polymerase to amplify or generatea specific piece of nucleic acid or to amplify or generate a specificpiece of nucleic acid which is complementary to a particular nucleicacid.

[0075] The “HRG tyrosine autophosphorylation assay” to detect thepresence or bioactivity of HRG ligands can be used to monitor thepurification of a ligand for the HER2 and HER3 receptors. This assay isbased on the assumption that a specific ligand for the receptor willstimulate autophosphorylation of the receptor, in analogy with EGF andits stimulation of EGF receptor autophosphorylation. See Sadich et al.,Anal. Biochem. 235:207-214 (1996). MDA-MB453 cells or MCF7 cells whichcontain high levels of p185^(HER2) receptors but negligible levels ofhuman EGF receptors, were obtained from the American Type CultureCollection, Rockville, Md. (ATCC No HTB-131) and maintained in tissueculture with 10% fetal calf serum in DMEM/Hams F12 (1:1) media. Forassay, the cells were trypsinized and plated at 150,000 cells/well in 24well dishes (Costar). After incubation with serum containing mediaovernight, the cells were placed in serum free media for 2-18 hoursbefore assay. Test samples of 100 uL aliquots were added to each well.The cells were incubated for 5-30 minutes (typically 30 min) at 37° C.and the media removed. The cells in each well were treated with 100 uLSDS gel denaturing buffer (SEPROSOL, Enpotech, Inc.) and the platesheated at e100C for 5 minutes to dissolve the cells and denature theproteins. Aliquots from each well were electrophoresed on 5-20% gradientSDS gels (NOVEX, Encinitas, Calif.) according to the manufacturer'sdirections. After the dye front reached the bottom of the gel, theelectrophoresis was terminated and a sheet of PVDF membrane (PROBLOTT,ABI) was placed on the gel and the proteins transferred from the gel tothe membrane in a blotting chamber (BioRad) at 200 mAmps for 30-60 min.After blotting, the membranes were incubated with TRIS buffered salinecontaining 0.1% TWEEN 20 detergent buffer with 5% BSA for 2-18 hrs toblock nonspecific binding, and then treated with a mouseanti-phosphotyrosine antibody (Upstate Biological Inc., N.Y.).Subsequently, the membrane blots were -treated with goat anti-mouseantibody conjugated to alkaline phosphatase. The gels were developedusing the PROTOBLOT System from Promega. After drying the membranes, thedensity of the bands corresponding to p185^(HER2) in each sample lanewas quantitated with a Hewlett Packard SCANJET Plus Scanner attached toa Macintosh computer. The number of receptors per cell in the MDA-MB-453cells is such that under these experimental conditions the p185^(HER2)receptor protein is the major protein which is labeled.

[0076] “Protein microsequencing” was accomplished based upon thefollowing procedures. Proteins from the final HPLC step were eithersequenced directly by automated Edman degradation with a model 470AApplied Biosystems gas phase sequencer equipped with a 120A PTH aminoacid analyzer or sequenced after digestion with various chemicals orenzymes. PTH amino acids were integrated using the CHROMPERFECT datasystem (Justice Innovations, Palo Alto, Calif.). Sequence interpretationwas performed on a VAX 11/785 Digital Equipment Corporation computer asdescribed (Henzel et al., J. Chromatography 404:41-52 (1987)). In somecases, aliquots of the HPLC fractions were electrophoresed on 5-20% SDSpolyacrylamide gels, electrotransferred to a PVDF membrane (PROBLOTT,ABI, Foster City, Calif.) and stained with Coomassie Brilliant Blue(Matsudaira, P., J. Biol. Chem. 262:10035-10038, 1987). The specificprotein was excised from the blot for N terminal sequencing. Todetermine internal protein sequences, HPLC fractions were dried undervacuum (SPEEDVAC), resuspended in appropriate buffers, and digested withcyanogen bromide, the lysine-specific enzyme Lys-C (Wako Chemicals,Richmond, Va.) or Asp-N (Boehringer Mannheim, Indianapolis, Ind.). Afterdigestion, the resultant peptides were sequenced as a mixture or wereresolved by HPLC on a C4 column developed with a propanol gradient in0.1% TFA before sequencing as described above.

[0077] “Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteinshaving the same structural characteristics. While antibodies exhibitbinding specificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

[0078] Papain digestion of antibodies produces two identicalantigen-binding fragments, called “Fab” fragments, each with a singleantigen-binding site, and a residual “Fc” fragment, whose name reflectsits ability to crystallize readily. Pepsin treatment yields an F(ab′)₂fragment that has two antigen-combining sites and is still capable ofcross-linking antigen.

[0079] “Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

[0080] The Fab fragment also contains the constant domain of the lightchain and the first constant domain (CH1) of the heavy chain. Fab′fragments differ from Fab fragments by the addition of a few residues atthe carboxyl terminus of the heavy chain CH1 domain including one ormore cysteine(s) from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

[0081] The “light chains” of antibodies (immunoglobulins) from anyvertebrate species can be assigned to one of two clearly distinct types,called kappa (κ) and lambda (λ), based on the amino acid sequences oftheir constant domains.

[0082] Depending on the amino acid sequence of the constant domain oftheir heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

[0083] The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), and antibody fragments so long as theyexhibit the desired biological activity.

[0084] “Antibody fragments” comprise a portion of a full lengthantibody, generally the antigen binding or variable domain thereof .Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.

[0085] The term “monoclonal antibody” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto conventional (polyclonal) antibody preparations which typicallyinclude different antibodies directed against different determinants(epitopes), each monoclonal antibody is directed against a singledeterminant on the antigen. The modifier “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKohler et al., Nature 256:495 (1975), or may be made by recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonalantibodies” may also be isolated from phage antibody libraries using thetechniques described in Clackson et al., Nature 352:624-628 (1991) andMarks et al., J. Mol. Biol. 222:581-597 (1991), for example.

[0086] The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

[0087] “Humanized” forms of non-human (e.g., murine) antibodies arechimeric antibodies which contain minimal sequence derived fromnon-human immunoglobulin. For the most part, humanized antibodies arehuman immunoglobulins (recipient antibody) in which hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.In some instances, framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable regions correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature 321:522-525 (1986); Reichmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992).

[0088] “Single-chain Fv” or “sFv” antibody fragments comprise the V_(H)and V_(L) domains of antibody, wherein these domains are present in asingle polypeptide chain. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, NewYork, pp. 269-315 (1994).

[0089] The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

[0090] The expression “linear antibodies” when used throughout thisapplication refers to the antibodies described in Zapata et al. ProteinEng. 8(10):1057-1062 (1995). Briefly, these antibodies comprise a pairof tandem Fd segments (V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific.

[0091] II. Use and Preparation of HRG Sequences

[0092] H. Preparation of HRG Sequences, Including Variants

[0093] The system to be employed in preparing HRG sequence will dependupon the particular HRG sequence selected. If the sequence issufficiently small HRG may be prepared by in vitro polypeptide syntheticmethods. Most commonly, however, HRG will be prepared in recombinantcell culture-using the host-vector systems described below. Suitable HRGincludes any biologically active and antigenetically active HRG.

[0094] In general, mammalian host cells will be employed, and such hostsmay or may not contain post-translational systems for processing HRGpreprosequences in the normal fashion. If the host cells contain suchsystems then it will be possible to recover natural subdomain fragmentssuch as HRG-GFD from the cultures. If not, then the proper processingcan be accomplished by transforming the hosts with the requiredenzyme(s) or by supplying them in an in vitro method. However, it is notnecessary to transform cells with the complete prepro or structuralgenes for a selected HRG when it is desired to only produce fragments ofHRG sequences such as an HRG-GFD. For example, a start codon is ligatedto the 5′ end of DNA encoding an HRG-GFD, this DNA is used to transformhost cells and the product expressed directly as the Met N-terminal form(if desired, the extraneous Met may be removed in vitro or by endogenousN-terminal demethionylases). Alternatively, HRG-GFD is expressed as afusion with a signal sequence recognized by the host cell, which willprocess and secrete the mature HRG-GFD as is further described below.Amino acid sequence variants of native HRG-GFD sequences are produced inthe same way.

[0095] HRG sequences located between the first N-terminal mature residueand the first N-terminal residue of HRG-GFD sequence, termed HRG-NTD,may function at least in part as an unconventional signal sequence or asa normally circulating carrier/precursor for HRG-GFD having uniquebiological activity. HRG-NTD is produced in the same fashion as the fulllength molecule but from expression of DNA in which a stop codon islocated at the C-terminus of HRG-NTD. In addition, HRG variants areexpressed from DNA encoding protein in which both the GFD and NTDdomains are in their proper orientation but which contain an amino acidinsertion, deletion or substitution at the GFD-NTD cleavage site(located within the sequence VKC) which inhibits or prevents proteolyticcleavage of the NTD-GFD joining site in vivo, and wherein a stop codonis positioned at the 3′ end of the GFD-encoding sequence. In an exampleof this group of variants (termed HRG-NTDXGFD), (1) the lysine residuefound in the NTD-GFD joining sequence VKC is deleted or (preferably)substituted by another residue other than arginyl such as histidyl,alanyl, threonyl or seryl and (2) a stop codon is introduced in thesequence RCT or RCQ in place of cysteinyl, or threonyl (for HRG-α) orglutaminyl (for HRG-β).

[0096] A preferred HRG-α ligand with binding affinity to p185^(HER2)comprises amino acids 226-265 of FIGS. 1A-D. This HRG-α ligand furthermay comprise up to an additional 1-20 amino acids preceding amino acid226 and 1-20 amino acids following amino acid 265. A preferred HRG-βligand with binding affinity to p185^(HER2) comprises amino acids226-265 of FIGS. 2A-E. This HRG-β ligand may comprise up to anadditional 1-20 amino acids preceding amino acid 226 and 1-20 aminoacids following amino acid 265.

[0097] As noted above, other HRG sequences to be prepared in accordancewith this invention are those of the GFD. These are synthesized in vitroor are produced in recombinant cell culture. These are produced mostinexpensively in yeast or E. coli by secretion under the control of aHRG-heterologous signal as described infra, although preparation inmammalian cells is also contemplated using a mammalian protein signalsuch as that of tPA, UK or a secreted viral protein. The GFD can be thesequence of a native HRG or may be a variant thereof as described below.GFD sequences include those in which one or more residues from a memberof the EGF family are substituted into or onto the GFD sequence.

[0098] An additional HRG is one which contains the GFD and the sequencebetween the C-terminus of GFD and the N-terminus of the transmembranedomain (the later being termed the C-terminal cleavage domain or CTC).In this variant (HRG-GFD-CTC) the DNA start codon is present at the 5′end of HRG-heterologous signal sequence or adjacent the 5′ end of theGFD-encoding region, and a stop codon is found in place of one of thefirst about 1 to 3 extra-cellular domain (ECD) residues or first about1-2 transmembrane region residues. In addition, in some HRG-GFD-CTCvariants the codons are modified in the GFD-CTC proteolysis site bysubstitution, insertion or deletion. The GFD-CTC proteolysis site is thedomain that contains the GFD C-terminal residue and about 5 residues N-and 5 residues C-terminal from this residue. It is known that Met-227terminal and Val-229 terminal HRG-A-GFD are biologically active. TheC-terminus for HRG-α-GFD may be Met-227, Lys-228, Val-229, Gln-230,Asn-231 or Gln-232, and for HRG-β-GFD may be Met-226, Ala-227, Ser-228,Phe-229, Trp-230, or Lys231/Ser231. The native C-terminus is determinedreadily by C-terminal sequencing, although it is not critical thatHRG-GFD have the native terminus so long as the GFD sequence possessesthe desired activity. In some embodiments of HRG-GFD-CTC variants, theamino acid change(s) in the CTC are screened for their ability to resistproteolysis in vitro and inhibit the protease responsible for generationof HRG-GFD.

[0099] HRG-ECD variants are made by providing a stop codon at the samelocation as for HRG-GFD-CTC variants. HRG-ECD may comprise any one ormore of the variants described above in connection with itssubfragments, e.g. the GFD-CTC variants containing CTC-proteolysis sitemodifications.

[0100] If it is desired to prepare the longer HRG polypeptides and the5′ or 3′ ends of the given HRG are not described herein, it may benecessary to prepare nucleic acids in which the missing domains aresupplied by homologous regions from more complete HRG nucleic acids.Alternatively, the missing domains can be obtained by probing librariesusing the DNAs disclosed in the Figures or fragments thereof.

A. Isolation of DNA Encoding Heregulin

[0101] The DNA encoding HRG may be obtained from any cDNA libraryprepared from tissue believed to possess HRG mRNA and to express it at adetectable level. HRG-A gene thus may be obtained from a genomiclibrary. Similar procedures may be used for the isolation of other HRG,such as HRG-β1, HRG-β2, or HRG-β3 encoding genes.

[0102] Libraries are screened with probes designed to identify the geneof interest or the protein encoded by it. For cDNA expression libraries,suitable probes include monoclonal or polyclonal antibodies thatrecognize and specifically bind to HRG-A; oligonucleotides of about20-80 bases in length that encode known or suspected portions of HRG-αcDNA from the same or different species; and/or complementary orhomologous cDNAs or fragments thereof that encode the same or a similargene. Appropriate probes for screening genomic DNA libraries include,but are not limited to, oligonucleotides; cDNAs or fragments thereofthat encode the same or a similar gene; and/or homologous genomic DNAsor fragments thereof. Screening the cDNA or genomic library with theselected probe may be conducted using standard procedures as describedin chapters 10-12 of Sambrook et al., supra.

[0103] An alternative means to isolate the gene encoding HRG-α is to usepolymerase chain reaction (PCR) methodology as described in section 14of Sambrook et al., supra. This method requires the use ofoligonucleotide probes that will hybridize to HRG-α. Strategies forselection of oligonucleotides are described below.

[0104] Another alternative method for obtaining the gene of interest isto chemically synthesize it using one of the methods described in Engelset al. (Agnew. Chem. Int Ed. Engl., 28: 716-734,1989), specificallyincorporated by reference. These methods include triester, phosphite,phosphoramidite and H-Phosphonate methods, PCR and other autoprimermethods, and oligonucleotide syntheses on solid supports. These methodsmay be used if the entire nucleic acid sequence of the gene is known, orthe sequence of the nucleic acid complementary to the coding strand isavailable, or alternatively, if the target amino acid sequence is known,one may infer potential nucleic acid sequences using known and preferredcoding residues for each amino acid residue.

[0105] A preferred method of practicing this invention is to usecarefully selected oligonucleotide sequences to screen cDNA librariesfrom various tissues, preferably human breast, colon, salivary gland,placental, fetal, brain, and carcinoma cell lines. Other biologicalsources of DNA encoding an heregulin-like ligand include other mammalsand birds. Among the preferred mammals are members of the followingorders: bovine, ovine, equine, murine, and rodentia.

[0106] The oligonucleotide sequences selected as probes should be ofsufficient length and sufficiently unambiguous that false positives areminimized. The actual nucleotide sequence(s) may, for example, be basedon conserved or highly homologous nucleotide a sequences or regions ofHRG-α. The oligonucleotides may be degenerate at one or more positions.The use of degenerate oligonucleotides may be of particular importancewhere a library is screened from a species in which preferential codonusage in that species is not known. The oligonucleotide must be labeledsuch that it can be detected upon hybridization to DNA in the librarybeing screened. The preferred method of labeling is to use ³²P-labeledATP with polynucleotide kinase, as is well known in the art, toradiolabel the oligonucleotide. However, other methods may be used tolabel the oligonucleotide, including, but not limited to, biotinylationor enzyme labeling.

[0107] Of particular interest is HRG-α nucleic acid that encodes afull-length polypeptide. In some preferred embodiments, the nucleic acidsequence includes the native HRG-α signal sequence. Nucleic acid havingall the protein coding sequence is obtained by screening selected cDNAor genomic libraries, and, if necessary, using conventional primerextension procedures as described in section 7.79 of Sambrook et al.,supra, to detect precursors and processing intermediates of mRNA thatmay not have been reverse-transcribed into cDNA.

[0108] HRG-α encoding DNA of FIGS. 1A-1D may be used to isolate DNAencoding the analogous ligand from other animal species viahybridization employing the methods discussed above. The preferredanimals are mammals, particularly bovine, ovine, equine, feline, canineand rodentia, and more specifically rats, mice and rabbits.

[0109] B. Amino Acid Sequence Variants of Herequlin

[0110] Amino acid sequence variants of HRG are prepared by introducingappropriate nucleotide changes into HRG DNA, or by in vitro synthesis ofthe desired HRG polypeptide. Such variants include, for example,deletions from, or insertions or substitutions of, residues within theamino acid sequence shown for human HRG sequences. Any combination ofdeletion, insertion, and substitution can be made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics. Excluded from the scope of this invention are HRGvariants or polypeptide sequences that are not novel and unobvious overthe prior art. The amino acid changes also may alter post-translationalprocesses of HRG-α, such as changing the number or position ofglycosylation sites, altering the membrane anchoring characteristics,altering the intra-cellular location of HRG by inserting, deleting, orotherwise affecting the leader sequence of the native HRG, or modifyingits susceptibility to proteolytic cleavage.

[0111] The HRG sequence may be proteolytically processed to create anumber of HRG fragments. HRG-GFD sequences of HRG-α all contain theamino acid sequence between HRG-α cysteine 226 and cysteine 265. Theamino terminus of HRG-A fragment may result from the cleavage of anypeptide bond between alanine 1 and cysteine 226, preferably adjacent toan arginine, lysine, valine, or methionine, and most preferably betweenmethionine 45 and serine 46. The carboxy terminus of HRG-α fragment mayresult from the cleavage of any peptide bond between cysteine 265,preferably adjacent to an arginine, lysine, valine, or methionine, andmost preferably between lysine 272 and valine 273, between lysine 278and alanine 279, or between lysine 285 and arginine 286. The resultingHRG-α ligands resulting from such proteolytic processing are thepreferred ligands.

[0112] HRG-β-GFD's are analogous to those discussed above forHRG-α-GFD's. Each HRG-β-GFD contains the polypeptide segment fromcysteine 212 to cysteine 251 of FIGS. 2A-E. The amino terminus of HRG-β1fragment may result from the cleavage of any peptide bond betweenalanine 1 and cysteine 212, preferably adjacent to an arginine, lysine,valine, or methionine, and most preferably between methionine 31 andserine 32. The carboxy terminus of HRG-β1 fragment may result from thecleavage of any peptide bond between cysteine 251 of FIGS. 2A-2E,preferably adjacent to an arginine, lysine, valine, or methionine, andmost preferably between valine 255 and methionine 256, between lysine261 and histidine 262, between lysine 276 and alanine 277, or betweenlysine 301 and thrionine 302. The resulting HRG-β1 ligands resultingfrom such proteolytic processing are among the preferred ligands.Similarly, processing to produce preferred fragment ligands of HRG-β2based upon the FIG. 3A-3E and HRG-β3 based upon FIGS. 4A-4C may beaccomplished by cleaving HRG sequences of FIGS. 3A-3E and 4A-4Cpreferably adjacent to an arginine, lysine, valine or methionine.

[0113] In designing amino acid sequence variants of HRG, the location ofthe mutation site and the nature of the mutation will depend on HRGcharacteristic(s) to be modified. The sites for mutation can be modifiedindividually or in series, e.g., by (1) substituting first withconservative amino acid choices and then with more radical selectionsdepending upon the results achieved, (2) deleting the target residue, or(3) inserting residues of other receptor ligands adjacent to the locatedsite.

[0114] A useful method for identification of certain residues or regionsof HRG polypeptide that are preferred locations for mutagenesis iscalled “alanine scanning mutagenesis” as described by Cunningham andWells (Science, 244: 1081-1085, 1989). Here, a residue or group oftarget residues are identified (e.g., charged residues such as arg, asp,his, lys, and glu) and replaced by a neutral or negatively charged aminoacid (most preferably alanine or polyalanine) to affect the interactionof the amino acids with the surrounding aqueous environment in oroutside the cell. Those domains demonstrating functional sensitivity tothe substitutions then are refined by introducing further or othervariants at or for the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined. For example, tooptimize the performance of a mutation at a given site, ala scanning orrandom mutagenesis may be conducted at the target codon or region andthe expressed HRG variants are screened for the optimal combination ofdesired activity.

[0115] There are two principal variables in the construction of aminoacid sequence variants: the location of the mutation site and the natureof the mutation. These are variants from HRG sequence, and may representnaturally occurring alleles (which will not require manipulation of HRGDNA) or predetermined mutant forms made by mutating the DNA, either toarrive at an allele or a variant not found in nature. In general, thelocation and nature of the mutation chosen will depend upon HRGcharacteristic to be modified. Obviously, such variations that, forexample, convert HRG into a known receptor ligand, are not includedwithin the scope of this invention, nor are any other HRG variants orpolypeptide sequences that are not novel and unobvious over the priorart.

[0116] Amino acid sequence deletions generally range from about 1 to 30residues, more preferably about 1 to 10 residues, and typically about 1to 5 are contiguous. Deletions may be introduced into regions of lowhomology with other EGF family precursors to modify the activity of HRG.Deletions from HRG in areas of substantial homology with other EGFfamily sequences will be more likely to modify the biological activityof HRG more significantly. The number of consecutive deletions will beselected so as to preserve the tertiary structure of HRG in the affecteddomain, e.g., cysteine crosslinking, beta-pleated sheet or alpha helix.

[0117] Amino acid sequence insertions include amino- and/orcarboxyl-terminal fusions ranging in length from one residue topolypeptides containing a hundred or more residues, as well asintrasequence insertions of single or multiple amino acid residues.Intrasequence insertions (i.e., insertions within HRG sequence) mayrange generally from about 1 to 10 residues, more preferably 1 to 5, andmost preferably 1 to 3. Examples of terminal insertions include HRG withan N-terminal methionyl residue (an artifact of the direct expression ofHRG in bacterial recombinant cell culture), and fusion of a heterologousN-terminal signal sequence to the N-terminus of HRG to facilitate thesecretion of mature HRG from recombinant host cells. Such signalsequences generally will be obtained from, and thus be homologous to,the intended host cell species. Suitable sequences include STII, tPA orIpp for E coli, alpha factor for yeast, and viral signals such as herpesgD for mammalian cells.

[0118] Other insertional variants of HRG include the fusion to the N- orC-terminus of HRG of an immunogenic polypeptide, e.g., bacterialpolypeptides such as beta-lactamase or an enzyme encoded by the E. colitrp locus, or yeast protein, bovine serum albumin, and chemotacticpolypeptides. C-terminal fusions of HRG-ECD with proteins having a longhalf-life such as immunoglobulin constant regions (or otherimmunoglobulin regions), albumin, or ferritin, as described in WO89102922, published 6 April 1989 are contemplated.

[0119] Another group of variants are amino acid substitution variants.These variants have at least one amino acid residue in HRG moleculeremoved and a different residue inserted in its place. The sites ofgreatest interest for substitutional mutagenesis include sitesidentified as the active site(s) of HRG, and sites where the amino acidsfound in HRG ligands from various species are substantially different interms of side-chain bulk, charge, and/or hydrophobicity. A likelysub-domain of HRG-GFD having biological activity as a growth factor isthe C-terminal segment, in particular within the sequence about fromglycine 218 to valine 226 (HRG-α), and glycine 218 to lysine 228/serine228 (HRG-β) based upon analogy to the EGF sub-sequence found to have EGFactivity.

[0120] Other sites of interest are those in which particular residues ofHRG-like ligands obtained from various species are identical. Thesepositions may be important for the biological activity of HRG. Thesesites, especially those falling within a sequence of at least threeother identically conserved sites, are substituted in a relativelyconservative manner. Such conservative substitutions are shown in Table1 under the heading of 3 “preferred substitutions”. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 1, oras further described below in reference to amino acid classes, areintroduced and the products screened. TABLE 1 Original ExemplaryPreferred Residue Substitutions Substitutions Ala (A) val; leu; ile valArg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu gluCys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro pro His (H)asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; leu norleucineLeu (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gln; asnarg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala leu Pro (P) glygly Ser (S) thr thr Thr (T) ser ser Trp (W) tyr tyr Tyr (Y) trp; phe;thr; ser phe Val (V) ile; leu; met; phe; ala; norleucine leu

[0121] Substantial modifications in function or immunological identityof HRG are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside chain properties:

[0122] 1) hydrophobic: norleucine, met, ala, val, leu, ile;

[0123] 2) neutral hydrophilic: cys, ser, thr;

[0124] 3) acidic: asp, glu;

[0125] 4) basic: asn, gin, his, lys, arg;

[0126] 5) residues that influence chain orientation: gly, pro; and

[0127] 6) aromatic: trp, tyr, phe.

[0128] Non-conservative substitutions will entail exchanging a member ofone of these classes for another. Such substituted residues may beintroduced into regions of HRG that are homologous with other receptorligands, or, more preferably, into the non-homologous regions of themolecule.

[0129] In one embodiment of the invention, it is desirable to inactivateone or more protease cleavage sites that are present in the molecule.These sites are identified by inspection of the encoded amino acidsequence. Where protease cleavage sites are identified, they arerendered inactive to proteolytic cleavage by substituting the targetedresidue with another residue, preferably a basic residue such asglutamine or a hydrophylic residue such as serine; by deleting theresidue; or by inserting a prolyl residue immediately after the residue.

[0130] In another embodiment, any methionyl residue other than thestarting methionyl residue of the signal sequence, or any residuelocated within about three residues N- or C-terminal to each suchmethionyl residue, is substituted by another residue (preferably inaccord with Table 1) or deleted. Alternatively, about 1-3 residues areinserted adjacent to such sites.

[0131] Any cysteine residues not involved in maintaining the properconformation of HRG also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking.

[0132] Sites particularly suited for substitutions, deletions orinsertions, or use as fragments, include, numbered from the N-terminusof HRG-α of FIGS. 1A-1D:

[0133] 1) potential glycosaminoglycan addition sites at theserine-glycine dipeptides at 42-43, 64-65, 151-152;

[0134] 2) potential asparagine-linked glycosylation at positions 164,170, 208 and 437, sites (NDS) 164-166, (NIT) 170-172, (NTS) 208-210, andNTS (609-611);

[0135] 3) potential O-glycosylation in a cluster of serine and threonineat 209-218;

[0136] 4) cysteines at 226, 234, 240, 254, 256 and 265;

[0137] 5) transmembrane domain at 287-309;

[0138] 6) loop 1 delineated by cysteines 226 and 240;

[0139] 7) loop 2 delineated by cysteines 234 and 254;

[0140] 8) loop 3 delineated by cysteines 256 and 265; and

[0141] 9) potential protease processing sites at 2-3, 8-9, 23-24, 33-34,36-37, 4546, 48-49, 62-63, 66-67,86-87,110-111, 123-124, 134-135,142-143, 272-273, 278-279 and 285-286;

[0142] Analogous regions in HRG-β1 may be determined by reference toits' sequence. The analogous HRG-β1 amino acids may be mutated ormodified as discussed above for HRG-A. Analogous regions in HRG-β2 mayalso be determined by reference to its' sequence. The analogous HRG-β2amino acids may be mutated or modified as discussed above for HRG-α orHRG-β1. Analogous regions in HRG-β3 may be determined by reference toits' sequence. Further, the analogous HRG-β3 amino acids may be mutatedor modified as discussed above for HRG-α, HRG-β1, or HRG-β2.

[0143] Another HRG variant is γ-HRG (or gamma-heregulin). γ-HRG is anypolypeptide sequence that possesses at least one biological property ofnative sequence γ-HRG having SEQ ID NO:11. The biological property ofthis variant is the same as for HRG noted above. This variantencompasses not only the polypeptide isolated from a native γ-HRG sourcesuch as human MDA-MB-175 cells or from another source, such as anotheranimal species, but also the polypeptide prepared by recombinant orsynthetic methods. It also includes variant forms including functionalderivatives, allelic variants, naturally occurring isoforms andanalogues thereof. Sometimes the γ-HRG is “native γ-HRG” which refers toendogenous γ-HRG polypeptide which has been isolated from a mammal. Theγ-HRG can also be “native sequence γ-HRG” insofar as it has the sameamino acid sequence as a native γ-HRG (e.g. human γ-HRG shown in FIGS.7A-7C). Amino acid sequence variants of the native sequence are preparedby introducing appropriate nucleotide changes into the native sequenceDNA, or by in vitro synthesis of the desired polypeptide. Such variantsinclude, for example, deletions from, or insertions or substitutions of,residues within the amino acid sequence shown for the human protein inFIGS. 7A-7C as generally described above for other HRG. Any combinationof deletion, insertion, and substitution is made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics. The amino acid changes also may alterpost-translational processes of the native sequence, such as changingthe number or position of O-linked glycosylation sites.

[0144] Another variant is the polypeptide referred to as sensory andmotor neuron derived factor (SMDF) whose nucleic acid and amino acidsequences (SEQ ID NOS:13 and 14) are shown in FIG. 8 which can beprepared as described in WO 96/15244. The SMDF polypeptides of theinvention exhibit the properties of binding to the HER2/HER3 receptorsand stimulating epithelial cell growth and differentiation in a mannersimilar to HRG polypeptides discussed above. Amino acid sequencevariants of native sequence SMDF are prepared by introducing appropriatenucleotide changes into the native sequence SMDF DNA, or by in vitrosynthesis of the desired SMDF polypeptide as observed generally abovefor other HRG. Such variants include, for example, deletions from, orinsertions or substitutions of, residues within the amino acid sequenceshown for human SMDF in FIG. 8. Any combination of deletion, insertion,and substitution is made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics. The aminoacid changes also may alter post-translational processes of the nativesequence SMDF, such as changing the number or position of O-linkedglycosylation sites.

[0145] Additional variants include polypeptides in which the variant hasan amino acid substitution at a selected residue corresponding to aresidue of 645-amino acid native human heregulin-β1 selected from:

[0146] S177, H178, L179, V180, K181, E184, E186,

[0147] K187, T188, V191, N192, G193, G194, E195,

[0148] M198, V199, K200, D201, N204, P205, S206,

[0149] R207, Y208, L209, K211, P213, N214, E215,

[0150] T217, G218, D219, Q222, N223, Y224, S228, and F229.

[0151] In a variation of this embodiment, the amino acid substitution isnot a replacement of the selected residue with an epidermal growthfactor (EGF) residue corresponding to the selected residue.

[0152] Other heregulin-β1 variants include an amino acid substitutionselected from:

[0153] S177W; H178S, E, R, or A; V180Q, I or E;

[0154] K181P orA; A183G; E184V, W, K, R, G, or N;

[0155] K185E, S, Q, or G;, E186R; K187E or A; T188Q;

[0156] E195Q; F197Y; M198R or K; K20OR; D201T or I;

[0157] P205T or Y; S206K, H, G, P, or R; R207Y;

[0158] Y208R or L; L209M or G; K211R; P213S, T, N, or K;

[0159] N214L, K, S, or E; F216M; N223H or W; and M2261.

[0160] In a varation of this embodiment, the heregulin variant includessets of amino acid substitutions selected from this group. Someheregulin variants of the invention having sets of amino acidsubstitutions exhibit at least a 50-fold increase in HER3 receptoraffinity, which is also accompanied by an increase in HER4 receptoraffinity. Specific variants include:

[0161] A183G, E184W, K185D, E186R, K187E, T188G, M226I;

[0162] A183D, E184K, K185S, E186R, K187E, T188G, M226I;

[0163] F197Y, M198K, K200R, D2011, M226I;

[0164] P205Y, S206G, R207Y, Y208L, L209M;

[0165] P205Y, S206R, R207Y, Y208R, L209M, M226I;

[0166] P205T, S206H, R207Y, Y208R, L209M;

[0167] P205T, S206K, R207Y, Y208R, L209G;

[0168] N223W, M226I;

[0169] N223H, M226I;

[0170] S177W, H178E, K181P, A183G, E184W, K185D, E186R, K187E,

[0171] T188G, M226I;

[0172] P205Y, S206G, R207Y, Y208L, L209M, M226I;

[0173] A183G, K185E, E186R, K187E, T188G, F197Y, M198R, D201T;

[0174] A183G, K185E, E186R, K187E, T188G, P205Y, S206G, R207Y, Y208L,L209M;

[0175] A183G, K185E, E186R, K187E, T188G, F197Y, M198R, D201T, P205Y,

[0176] S206G, R207Y, Y208L, L209M;

[0177] A183G, K185E, E186R, K187E, T188G, M226I; sF197Y, M198R, D201T,P205Y, S206G, R207Y, Y208L, L209M;

[0178] F197Y, M198R, D201T, P205Y, S206G, R207Y, Y208L, L209M, M226I;

[0179] F197Y, M198R, D201T, M226I;

[0180] A183G, K185E, E186R, K187E, T188G, F197Y, M198R, D201T, M226I;

[0181] A183G, K185E, E186R, K187E, T188G, P205Y, S206G, R207Y, Y208L,L209M, M226I;

[0182] A183G, K185E, E186R, K187E, T188G, F197Y, M198R, D201T, P205Y,S206G, R207Y, Y208L, L209M, M226I;

[0183] F197Y, M198R, D201T, P205Y, S206G, R207Y, Y208L, L209M, N223H,M2261; and

[0184] A183G, K185E, E186R, K187E, T188G, F197Y, M198R, D201T, P205Y,S206G, R207Y, Y208L, L209M, N223H, M2261.

[0185] In addition to including one or more of the amino acidsubstitutions disclosed herein, the heregulin variant can have one ormore other modifications, such as an amino acid substitution, aninsertion of at least one amino acid, a deletion of at least one aminoacid, or a chemical modification. For example, the invention provides aheregulin variant that is a fragment. In a variation of this embodiment,the fragment includes residues corresponding to a portion of humanheregulin-p1 extending from about residue 175 to about residue 230(i.e., the EGF-like domain). For example, the fragment can extend fromresidue 177 to residue 244 and may be prepared by recombinant techniques(rHRGβ1-177-244).

[0186] DNA encoding amino acid sequence variants of HRG is prepared by avariety of methods known in the art. These methods include, but are notlimited to, isolation from a natural source (in the case of naturallyoccurring amino acid sequence variants) or preparation byoligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, and cassette mutagenesis of an earlier prepared variant ora non-variant version of HRG. These techniques may utilize HRG nucleicacid (DNA or RNA), or nucleic acid complementary to HRG nucleic acid.

[0187] Oligonucleotide-mediated mutagenesis is a preferred method forpreparing substitution, deletion, and insertion variants of HRG DNA.This technique is well known in the art as described by Adelman et al.,DNA, 2: 183 (1983). Briefly, HRG DNA is altered by hybridizing anoligonucleotide encoding the desired mutation to a DNA template, wherethe template is the single-stranded form of a plasmid or bacteriophagecontaining the unaltered or native DNA sequence of HRG. Afterhybridization, a DNA polymerase is used to synthesize an entire secondcomplementary strand of the template that will thus incorporate theoligonucleotide primer, and will code for the selected alteration in HRGDNA.

[0188] Generally, oligonucleotides of at least 25 nucleotides in lengthare used. An optimal oligonucleotide will have 12 to 15 nucleotides thatare completely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al. (Proc.Nat!. Acad. Sci. USA, 75: 5765,1978).

[0189] Single-stranded DNA template may also be generated by denaturingdouble-stranded plasmid (or other) DNA using standard techniques.

[0190] For alteration of the native DNA sequence (to generate amino acidsequence variants, for example), the oligonucleotide is hybridized tothe single-stranded template under suitable hybridization conditions. ADNA polymerizing enzyme, usually the Klenow fragment of DNA polymeraseI, is then added to synthesize the complementary strand of the templateusing the oligonucleotide as a primer for synthesis. A heteroduplexmolecule is thus formed such that one strand of DNA encodes the mutatedform of HRG, and the other strand (the original template) encodes thenative, unaltered sequence of HRG. This heteroduplex molecule is thentransformed into a suitable host cell, usually a prokaryote such as E.coli JM101. After the cells are grown, they are plated onto agaroseplates and screened using the oligonucleotide primer radiolabeled with³²P-phosphate to identify the bacterial colonies that contain themutated DNA. The mutated region is then removed and placed in anappropriate vector for protein production, generally an expressionvector of the type typically employed for transformation of anappropriate host.

[0191] The method described immediately above may be modified such thata homoduplex molecule is created wherein both strands of the plasmidcontain the mutation(s). The modifications are as follows: thesingle-stranded oligonucleotide is annealed to the single-strandedtemplate as described above. A mixture of three deoxyribonucleotides,deoxydiboadenosine (dATP), deoxyriboguanosine (dGTP), anddeoxyribothymidine (dTTP), is combined with a modifiedthio-deoxyribocytosine called dCTP-(aS) (which can be obtained fromAmersham Corporation). This mixture is added to thetemplate-oligonucleotide complex. Upon addition of DNA polymerase tothis mixture, a strand of DNA identical to the template except for themutated bases is generated. In addition, this new strand of DNA willcontain dCTP-(aS) instead of dCTP, which serves to protect it fromrestriction endonuclease digestion. After the template strand of thedouble-stranded heteroduplex is nicked with an appropriate restrictionenzyme, the template strand can be digested with ExoIII nuclease oranother appropriate nuclease past the region that contains the site(s)to be mutagenized. The reaction is then stopped to leave a molecule thatis only partially single-stranded. A complete double-stranded DNAhomoduplex is then formed using DNA polymerase in the presence of allfour deoxyribonucleotide triphosphates, ATP, and DNA ligase. Thishomoduplex molecule can then be transformed into a suitable host cellsuch as E. coli JM101, as described above.

[0192] DNA encoding HRG mutants with more than one amino acid to besubstituted may be generated in one of several ways. If the amino acidsare located close together in the polypeptide chain, they may be mutatedsimultaneously using one oligonucleotide that codes for all of thedesired amino acid substitutions. If, however, the amino acids arelocated some distance from each other (separated by more than about tenamino acids), it is more difficult to generate a single oligonucleotidethat encodes all of the desired changes. Instead, one of two alternativemethods may be employed.

[0193] In the first method, a separate oligonucleotide is generated foreach amino acid to be substituted. The oligonucleotides are thenannealed to the single-stranded template DNA simultaneously, and thesecond strand of DNA that is synthesized from the template will encodeall of the desired amino acid substitutions.

[0194] The alternative method involves two or more rounds of mutagenesisto produce the desired mutant. The first round is as described for thesingle mutants: wild-type DNA is used for the template, anoligonucleotide encoding the first desired amino acid substitution(s) isannealed to this template, and the heteroduplex DNA molecule is thengenerated. The second round of mutagenesis utilizes the mutated DNAproduced in the first round of mutagenesis as the template. Thus, thistemplate already contains one or more mutations. The oligonucleotideencoding the additional desired amino acid substitution(s) is thenannealed to this template, and the resulting strand of DNA now encodesmutations from both the first and second rounds of mutagenesis. Thisresultant DNA can be used as a template in a third round of mutagenesis,and so on.

[0195] PCR mutagenesis is also suitable for making amino acid variantsof HRG. While the following discussion refers to DNA, it is understoodthat the technique also finds application with RNA. The PCR techniquegenerally refers to the following procedure (see Erlich, supra, thechapter by R. Higuchi, p. 61-70). When small amounts of template DNA areused as starting material in a PCR, primers that differ slightly insequence from the corresponding region in a template DNA can be used togenerate relatively large quantities of a specific DNA fragment thatdiffers from the template sequence only at the positions where theprimers differ from the template. For introduction of a mutation into aplasmid DNA, one of the primers is designed to overlap the position ofthe mutation and to contain the mutation; the sequence of the otherprimer must be identical to a stretch of sequence of the opposite strandof the plasmid, but this sequence can be located anywhere along theplasmid DNA. It is preferred, however, that the sequence of the secondprimer is located within 200 nucleotides from that of the first, suchthat in the end the entire amplified region of DNA bounded by theprimers can be easily sequenced. PCR amplification using a primer pairlike the one just described results in a population of DNA fragmentsthat differ at the position of the mutation specified by the primer, andpossibly at other positions, as template copying is somewhaterror-prone.

[0196] If the ratio of template to product material is extremely low,the vast majority of product DNA fragments incorporate the desiredmutation(s). This product material is used to replace the correspondingregion in the plasmid that served as PCR template using standard DNAtechnology. Mutations at separate positions can be introducedsimultaneously by either using a mutant second primer, or performing asecond PCR with different mutant primers and ligating the two resultingPCR fragments simultaneously to the vector fragment in a three (ormore)-part ligation.

[0197] In a specific example of PCR mutagenesis, template plasmid DNA (1mg) is linearized by digestion with a restriction endonuclease that hasa unique recognition site in the plasmid DNA outside of the region to beamplified. Of this material, 100 ng is added to a PCR mixture containingPCR buffer, which contains the four deoxynucleotide tri-phosphates andis included in the GENEAMP kits (obtained from Perkin-Elmer Cetus,Norwalk, Conn. and Emeryville, Calif.), and 25 pmole of eacholigonucleotide primer, to a final volume of 50 ml. The reaction mixtureis overlaid with 35 ml mineral oil. The reaction is denatured for 5minutes at 100° C., placed briefly on ice, and then 1 ml Thermusaquaticus (Taq) DNA polymerase (5 units/ml, purchased from Perkin-ElmerCetus, Norwalk, Conn. and Emeryville, Calif.) is added below the mineraloil layer. The reaction mixture is then inserted into a DNA ThermalCycler (purchased from Perkin-Elmer Cetus) programmed as follows:

[0198] 2 min. 55° C.,

[0199] 30 sec. 72° C., then 19 cycles of the following:

[0200] 30 sec. 94° C.,

[0201] 30 sec. 55° C., and

[0202] 30 sec. 72° C.

[0203] At the end of the program, the reaction vial is removed from thethermal cycler and the aqueous phase transferred to a new vial,extracted with phenol/chloroform (50:50:vol), and ethanol precipitated,and the DNA is recovered by standard procedures. This material issubsequently subjected to the appropriate treatments for insertion intoa vector.

[0204] Another method for preparing variants, cassette mutagenesis, isbased on the technique described by Wells et al. (Gene, 34: 315,1985).The starting material is the plasmid (or other vector) comprising HRGDNA to be mutated. The codon(s) in HRG DNA to be mutated are identified.There must be a unique restriction endonuclease site on each side of theidentified mutation site(s). If no such restriction sites exist, theymay be generated using the above-described oligonucleotide-mediatedmutagenesis method to introduce them at appropriate locations in HRGDNA. After the restriction sites have been introduced into the plasmid,the plasmid is cut at these sites to linearize it. A double-strandedoligonucleotide encoding the sequence of the DNA between the restrictionsites but containing the desired mutation(s) is synthesized usingstandard procedures. The two strands are synthesized separately and thenhybridized together using standard techniques. This double-strandedoligonucleotide is referred to as the cassette. This cassette isdesigned to have 3′ and 5′ ends that are compatible with the ends of thelinearized plasmid, such that it can be directly ligated to the plasmid.This plasmid now contains the mutated HRG DNA sequence.

[0205] C. Insertion of DNA into a Cloning Vehicle

[0206] The cDNA or genomic DNA encoding native or variant HRG isinserted into a replicable vector for further cloning (amplification ofthe DNA) or for expression. Many vectors are available, and selection ofthe appropriate vector will depend on 1) whether it is to be used forDNA amplification or for DNA expression, 2) the size of the DNA to beinserted into the vector, and 3) the host cell to be transformed withthe vector. Each vector contains various components depending on itsfunction (amplification of DNA or expression of DNA) and the host cellfor which it is compatible. The vector components generally include, butare not limited to, one or more of the following: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence.

[0207] (i) Signal Sequence Component

[0208] In general, the signal sequence may be a component of the vector,or it may be a part of HRG DNA that is inserted into the vector. Thenative HRG DNA is believed to encode a signal sequence at the aminoterminus (5′ end of the DNA encoding HRG) of the polypeptide that iscleaved during post-translational processing of the polypeptide to formthe mature HRG polypeptide ligand that binds to the HER2/HER3 receptor,although a conventional signal structure is not apparent. Native HRG is,secreted from the cell but remains lodged in the membrane because itcontains a transmembrane domain and a cytoplasmic region in the carboxylterminal region of the polypeptide. Thus, in a secreted, soluble versionof HRG the carboxyl terminal domain of the molecule, including thetransmembrane domain, is ordinarily deleted. This truncated variant HRGpolypepfide may be secreted from the cell, provided that the DNAencoding the truncated variant encodes a signal sequence recognized bythe host.

[0209] HRG of this invention may be expressed not only directly, butalso as a fusion with a heterologous polypeptide, preferably a signalsequence or other polypeptide having a specific cleavage site at the N-and/or C-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofHRG DNA that is inserted into the vector. Included within the scope ofthis invention are HRG with the native signal sequence deleted andreplaced with a heterologous signal sequence. The heterologous signalsequence selected should be one that is recognized and processed, i.e.,cleaved by a signal peptidase, by the host cell. For prokaryotic hostcells that do not recognize and process the native HRG signal sequence,the signal sequence is substituted by a prokaryotic signal sequenceselected, for example, from the group of the alkaline phosphatase,penicillinase, Ipp, or heat-stable enterotoxin II leaders. For yeastsecretion the native HRG signal sequence may be substituted by the yeastinvertase, alpha factor, or acid phosphatase leaders. In mammalian cellexpression the native signal sequence is satisfactory, although othermammalian signal sequences may be suitable.

[0210] (ii) Origin of Replication Component

[0211] Both expression and cloning vectors generally contain a nucleicacid sequence that enables the vector to replicate in one or moreselected host cells. Generally, in cloning vectors this sequence is onethat enables the vector to replicate independently of the hostchromosomal DNA, and includes origins of replication or autonomouslyreplicating sequences. Such sequences are well known for a variety ofbacteria, yeast, and viruses. The origin of replication from the plasmidpBR322 is suitable for most Gram-negative bacteria, the 2 m plasmidorigin is suitable for yeast, and various viral origins (SV40, polyoma,adenovirus, VSV or BPV) are useful for cloning vectors in mammaliancells. Generally, the origin of replication component is not needed formammalian expression vectors (the SV40 origin may typically be used onlybecause it contains the early promoter).

[0212] Most expression vectors are “shuttle” vectors, i.e., they arecapable of replication in at least one class of organisms but can betransfected into another organism for expression. For example, a vectoris cloned in E. coli and then the same vector is transfected into yeastor mammalian cells for expression even though it is not capable ofreplicating independently of the host cell chromosome.

[0213] DNA may also be amplified by insertion into the host genome. Thisis readily accomplished using Bacillus species as hosts, for example, byincluding in the vector a DNA sequence that is complementary to asequence found in Bacillus genomic DNA. Transfection of Bacillus withthis vector results in homologous recombination with the genome andinsertion of HRG DNA. However, the recovery of genomic DNA encoding HRGis more complex than that of an exogenously replicated vector becauserestriction enzyme digestion is required to excise HRG DNA. DNA can beamplified by PCR and directly transfected into the host cells withoutany replication component.

[0214] (iii) Selection Gene Component

[0215] Expression and cloning vectors should contain a selection gene,also termed a selectable marker. This gene encodes a protein necessaryfor the survival or growth of transformed host cells grown in aselective culture medium. Host cells not transformed with the vectorcontaining the selection gene will not survive in the culture medium.Typical selection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli.

[0216] One example of a selection scheme utilizes a drug to arrestgrowth of a host cell. Those cells that are successfully transformedwith a heterologous gene express a protein conferring drug resistanceand thus survive the selection regimen. Examples of such dominantselection use the drugs neomycin (Southern et al., J. Molec. Appl.Genet. 1: 327,1982), mycophenolic acid (Mulligan et al., Science 209:1422,1980) or hygromycin (Sugden et al., Mol. Cell. Biol. 5:410413,1985). The three examples given above employ bacterial genesunder eukaryotic control to convey resistance to the appropriate drugG418 or neomycin (geneticin), xgpt (mycophenolic acid), or hygromycin,respectively.

[0217] Another example of suitable selectable markers for mammaliancells are those that enable the identification of cells competent totake up HRG nucleic acid, such as dihydrofolate reductase (DHFR) orthymidine kinase. The mammalian cell transformants are placed underselection pressure which only the transformants are uniquely adapted tosurvive by virtue of having taken up the marker. Selection pressure isimposed by culturing the transformants under conditions in which theconcentration of selection agent in the medium is successively changed,thereby leading to amplification of both the selection gene and the DNAthat encodes HRG. Amplification is the process by which genes in greaterdemand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Increased quantities of HRG are synthesized from theamplified DNA.

[0218] For example, cells transformed with the DHFR selection gene arefirst identified by culturing all of the transformants in a culturemedium that contains methotrexate (Mtx), a competitive antagonist ofDHFR. An appropriate host cell when wild-type DHFR is employed is theChinese hamster ovary (CHO) cell line deficient in DHFR activity,prepared and propagated as described by Urlaub and Chasin, Proc. Natl.Acad. Sci. USA, 77: 4216, 1980. The transformed cells are then exposedto increased levels of methotrexate. This leads to the synthesis ofmultiple copies of the DHFR gene, and, concomitantly, multiple copies ofother DNA comprising the expression vectors, such as the DNA encodingHRG. This amplification technique can be used with any otherwisesuitable host, e.g., ATCC No. CCL61 CHO-KL, notwithstanding the presenceof endogenous DHFR if, for example, a mutant DHFR gene that is highlyresistant to Mtx is employed (EP 117,060). Alternatively, host cells(particularly wild-type hosts that contain endogenous DHFR) transformedor co-transformed with DNA sequences encoding HRG, wild-type DHFRprotein, and another selectable marker such as aminoglycoside 3′phosphotransferase (APH) can be selected by cell growth in mediumcontaining a selection agent for the selectable marker such as anaminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418 (see U.S.Pat. No. 4,965,199).

[0219] A suitable selection gene for use in yeast is the trpl genepresent in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39,1979; Kingsman et al., Gene, 7: 141, 1979; or Tschemper et al., Gene,10: 157, 1980). The trp1 gene provides a selection marker for a mutantstrain of yeast lacking the ability to grow in tryptophan, for example,ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85: 12, 1977). The presenceof the trpl lesion in the yeast host cell genome then provides aneffective environment for detecting transformation by growth in theabsence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC20,622 or 38,626) are complemented by known plasmids bearing the Leu2gene.

[0220] (iv) Promoter Component

[0221] Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to HRG nucleicacid. Promoters are untranslated sequences located upstream (5′) to thestart codon of a structural gene (generally within about 100 to 1000 bp)that control the transcription and translation of a particular nucleicacid sequence, such as HRG to which they are operably linked. Suchpromoters typically fall into two classes, inducible and constitutive.Inducible promoters are promoters that initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, e.g., the presence or absence of a nutrient or achange in temperature. At this time a large number of promotersrecognized by a variety of potential host cells are well known. Thesepromoters are operably linked to DNA encoding HRG by removing thepromoter from the source DNA by restriction enzyme digestion andinserting the isolated promoter sequence into the vector. Both thenative HRG promoter sequence and many heterologous promoters may be usedto direct amplification and/or expression of HRG DNA. However,heterologous promoters are preferred, as they generally permit greatertranscription and higher yields of expressed HRG as compared to thenative HRG promoter.

[0222] Promoters suitable for use with prokaryotic hosts include theb-lactamase and lactose promoter systems (Chang et al., Nature, 275:615, 1978; and Goeddel et al., Nature 281: 544, 1979), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic AcidsRes., 8: 4057, 1980 and EP 36,776), tPA (U.S. Pat. No. 5,641,655) andhybrid promoters such as the tac promoter (deBoer et al., Proc. Natl.Acad. Sci. USA 80: 21-25, 1983). However, other known bacterialpromoters are suitable. Their nucleotide sequences have been published,thereby enabling a skilled worker operably to ligate them to DNAencoding HRG (Siebenlist et al., Cell 20: 269, 1980) using linkers oradaptors to supply any required restriction sites. Promoters for use inbacterial systems also generally will contain a Shine-Dalgarno (S.D.)sequence operably linked to the DNA encoding HRG.

[0223] Suitable promoting sequences for use with yeast hosts include thepromoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem., 255: 2073, 1980) or other glycolytic enzymes (Hess et al., J.Adv. Enzyme Reg 7: 149, 1968; and Holland, Biochemistry 17: 4900, 1978),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

[0224] Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin Hitzeman et al., EP 73,657A. Yeast enhancers also are advantageouslyused with yeast promoters.

[0225] Promoter sequences are known for eukaryotes. Virtually alleukaryotic genes have an AT-rich region located approximately 25 to 30bases upstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCMT region where X may be any nucleotide. At the 3′end of most eukaryotic genes is an MTAAA sequence that may be the signalfor addition of the poly A tail to the 3′ end of the coding sequence.All of these sequences are suitably inserted into mammalian expressionvectors.

[0226] HRG gene transcription from vectors in mammalian host cells maybe controlled by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504, published Jul. 5, 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and mostpreferably Simian Virus 40 (SV40), from heterologous mammalianpromoters, e.g., the actin promoter or an immunoglobulin promoter, fromheat-shock promoters, and from the promoter normally associated with HRGsequence, provided such promoters are compatible with the host cellsystems.

[0227] The early and late promoters of the SV40 virus are convenientlyobtained as an SV40 restriction fragment that also contains the SV40viral origin of replication (Fiers et al., Nature, 273:113 (1978);Mulligan and Berg, Science, 209: 1422-1427 (1980); Pavlakis et al.,Proc. Natl. Acad. Sci. USA, 78: 7398-7402 (1981)). The immediate earlypromoter of the human cytomegalovirus is conveniently obtained as aHindIII E restriction fragment (Greenaway et al., Gene, 18: 355-360(1982)). A system for expressing DNA in mammalian hosts using the bovinepapilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. Amodification of this system is described in U.S. Pat. No. 4,601,978. Seealso Gray et al., Nature, 295: 503-508 (1982) on expressing cDNAencoding immune interferon in monkey cells; Reyes et al., Nature, 297:598-601 (1982) on expression of human b-interferon cDNA in mouse cellsunder the control of a thymidine kinase promoter from herpes simplexvirus; Canaani and Berg, Proc. Natl. Acad. Sci. USA, 79: 5166-5170(1982) on expression of the human interferon b1 gene in cultured mouseand rabbit cells; and Gorman et al., Proc. Natl. Acad. Sci. USA, 79:6777-6781 (1982) on expression of bacterial CAT sequences in CV-1 monkeykidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells,HeLa cells, and mouse NIH-3T3 cells using the Rous sarcoma virus longterminal repeat as a promoter.

[0228] (v) Enhancer Element Component

[0229] Transcription of a DNA encoding HRG of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from10-300 bp, that act on a promoter to increase its transcription.Enhancers are relatively orientation and position independent havingbeen found 5′ (Laimins et al., Proc. Natl. Acad. Sci. USA, 78: 993,1981) and 3′ (Lusky et al., Mol. Cell Bio., 3: 1108, 1983) to thetranscription unit, within an intron (Banerji et al., Cell, 33: 729,1983) as well as within the coding sequence itself (Osborne et al., Mol.Cell Bio., 4: 1293, 1984). Many enhancer sequences are now known frommammalian genes (globin, elastase, albumin, a-fetoprotein and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers (see also Yaniv, Nature, 297: 17-18(1982)) on enhancing elements for activation of eukaryotic promoters.The enhancer may be spliced into the vector at a position 5′ or 3′ toHRG DNA, but is preferably located at a site 5′ from the promoter.

[0230] (vi) Transcription Termination Component

[0231] Expression vectors used in eukaryotic host cells (yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding HRG. The 3′ untranslatedregions also include transcription termination sites.

[0232] Construction of suitable vectors containing one or more of theabove listed components the desired coding and control sequences employsstandard ligation techniques. Isolated plasmids or DNA fragments arecleaved, tailored, and religated in the form desired to generate theplasmids required.

[0233] For analysis to confirm correct sequences in plasmidsconstructed, the ligation mixtures are used to transform E. coli K12strain 294 (ATCC 31,446) and successful transformants selected byampicillin or tetracycline resistance where appropriate. Plasmids fromthe transformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res. 9: 309 (1981) or by the method of Maxam et al., Methods inEnzymology 65: 499 (1980).

[0234] Particularly useful in the practice of this invention areexpression vectors that provide for the transient expression inmammalian cells of DNA encoding HRG. In general, transient expressioninvolves the use of an expression vector that is able to replicateefficiently in a host cell, such that the host cell accumulates manycopies of the expression vector and, in turn, synthesizes high levels ofa desired polypeptide encoded by the expression vector. Transientexpression systems, comprising a suitable expression vector and a hostcell, allow for the convenient positive identification of polypeptidesencoded by cloned DNAs, as well as for the rapid screening of suchpolypeptides for desired biological or physiological properties. Thus,transient expression systems are particularly useful in the inventionfor purposes of identifying analogs and variants of HRG that haveHRG-like activity. Such a transient expression system is described inU.S. PAt. No. 5,024,939.

[0235] Other methods, vectors, and host cells suitable for adaptation tothe synthesis of HRG in recombinant vertebrate cell culture aredescribed in Gething et al., Nature 293: 620-625, 1981; Mantei et al.,Nature, 281: 4046,1979; Levinson et al., EP 117,060 and EP 117,058. Aparticularly useful expression plasmid for mammalian cell cultureexpression of HRG is pRK5 (EP pub. no. 307,247) or pSVI6B (U.S. Ser. No.071441,574, filed 22 November 1989, the disclosure of which isincorporated herein by reference).

[0236] D. Selection and Transformation of Host Cells

[0237] Suitable host cells for cloning or expressing the vectors hereinare the prokaryote, yeast, or higher eukaryote cells described above.Suitable prokaryotes include eubacteria, such as Gram-negative orGram-positive organisms, for example, E. coli, Bacilli such as B.subtilis, Pseudomonas species such as P. aeruginosa, Salmonellatyphimurium, or Serratia marcescans. One preferred E. coli cloning hostis E. coli 294 (ATCC 31,446), although other strains such as E. coli B,E. coli _(x)1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) aresuitable. These examples are illustrative rather than limiting.Preferably the host cell should secrete minimal amounts of proteolyticenzymes. Alternatively, in vitro methods of cloning, e.g., PCR or othernucleic acid polymerase reactions, are suitable.

[0238] In addition to prokaryotes, eukaryotic microbes such asfilamentous fungi or yeast are suitable hosts for HRG-encoding vectors.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among lower eukaryotic host microorganisms. However, a number ofother genera, species, and strains are commonly available and usefulherein, such as Schizosaccharomyces pombe (Beach and Nurse, Nature, 290:140 (1981); EP 139,383, published May 2, 1985), Kluyveromyces hosts(U.S. Ser. No. 4,943,529) such as, e.g., K. lactis (Louvencourt et al.,J. Bacterio!., 737 (1983); K. fragilis, K. bulgaricus, K.thermotolerans, and K. marxianus, yarrowia (EP 402,226); Pichia pastoris(EP 183,070), Sreekrishna et al., J. Basic Microbiol., 28: 265-278(1988); Candida, Trichoderma reesia (EP 244,234); Neurospora crassa(Case et al., Proc. Natl. Acad. Sci. USA, 76: 5259-5263 (1979), andfilamentous fungi such as, e.g, Neurospora, Penicillium, Tolypocladium(WO 91/00357, published Jan. 10, 1991), and Aspergillus hosts such as A.nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112: 284-289(1983); Tilburn et al., Gene, 26: 205-221 (1983); Yelton et al., Proc.Natl. Acad. Sci. USA, 81: 1470-1474 (1984) and A. niger (Kelly andHynes, EMBO J., 4: 475479 (1985)).

[0239] Suitable host cells for the expression of glycosylated HRGpolypeptide are derived from multicellular organisms. Such host cellsare capable of complex processing and glycosylation activities. Inprinciple, any higher eukaryotic cell culture is workable, whether fromvertebrate or invertebrate culture. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruiffly), and Bombyxmori host cells have been identified (see, e.g., Luckow et al.,Bio/Technology, 6: 47-55 (1988); Miller et al., in Genetic Engineering,Setlow, J. K. et al., eds., Vol. 8 (Plenum Publishing, 1986), pp.277-279; and Maeda et al., Nature, 315: 592-594 (1985)). A variety ofsuch viral strains are publicly available, e.g., the L-1 variant ofAutographa californica NPV and the Bm-5 strain of Bombyx mori NPV, andsuch viruses may be used as the virus herein according to the presentinvention, particularly for transfection of Spodoptera frugiperda cells.Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens, which has been previously manipulated tocontain HRG DNA. During incubation of the plant cell culture with A.tumefaciens, the DNA encoding HRG is transferred to the plant cell hostsuch that it is transfected, and will, under appropriate conditions,express HRG DNA. In addition, regulatory and signal sequences compatiblewith plant cells are available, such as the nopaline synthase promoterand polyadenylation signal sequences (Depicker et al., J. Mol. Appl.Gen., 1: 561 (1982)). In addition, DNA segments isolated from theupstream region of the T-DNA 780 gene are capable of activating orincreasing transcription levels of plant-expressible genes inrecombinant DNA-containing plant tissue (see EP 321,196, published Jun.21, 1989).

[0240] However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) has become aroutine procedure in recent years (Tissue Culture, Academic Press, Kruseand Patterson, editors (1973)). Examples of useful mammalian host celllines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36: 59,1977); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,77: 4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (Wl38,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y Acad.Sci., 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomacell line (Hep G2). Preferred host cells are human embryonic kidney 293and Chinese hamster ovary cells.

[0241] Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors of this invention andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

[0242] Transfection refers to the taking up of an expression vector by ahost cell whether or not any coding sequences are in fact expressed.Numerous methods of transfection are known to the ordinarily skilledartisan, for example, CaPO₄ and electroporation. Successful transfectionis generally recognized when any indication of the operation of thisvector occurs within the host cell.

[0243] Transformation means introducing DNA into an organism so that theDNA is replicable, either as an extrachromosomal element or bychromosomal integration. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride, as described in section 1.82 ofSambrook et al., supra, is generally used for prokaryotes or other cellsthat contain substantial cell-wall barriers. Infection withAgrobacterium tumefaciens is used for transformation of certain plantcells, as described by Shaw et al, Gene, 23: 315 (1983) and WO 89/05859,published June 29, 1989. For mammalian cells without such cell walls,the calcium phosphate precipitation method described in sections16.30-16.37 of Sambrook et al, supra, is preferred. General aspects ofmammalian cell host system transformations have been described by Axelin U.S. Pat. No. 4,399,216, issued Aug. 16, 1983. Transformations intoyeast are typically carried out according to the method of Van Solingenet al., J. Bact., 130: 946 (1977) and Hsiao et al., Proc. Natl. Acad.Sci. (USA), 76: 3829 (1979). However, other methods for introducing DNAinto cells such as by nuclear injection, electroporation, or protoplastfusion may also be used.

[0244] E. Culturing the Host Cells

[0245] Prokaryotic cells used to produce HRG polypeptide of thisinvention are cultured in suitable media as described generally inSambrook et al., supra.

[0246] The mammalian host cells used to produce HRG of this inventionmay be cultured in a variety of media. Commercially available media suchas Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham and Wallace, Meth. Enz., 58: 44 (1979), Barnes andSato, Anal Biochem., 102: 255 (1980), U.S. Pat. Nos. 4,767,704;4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195; U.S. Pat.Re. No. 30,985; U.S. Pat. No. 5,122,469 or U.S. Ser. No. 07/592,141,filed on Oct. 3, 1990, the disclosures of all of which are incorporatedherein by reference, may be used as culture media for the host cells.Any of these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleosides (such as adenosine andthymidine), antibiotics (such as GENTAMYCIN drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

[0247] The host cells referred to in this disclosure encompass cells inin vitro culture as well as cells that are within a host animal.

[0248] It is further envisioned that HRG of this invention may beproduced by homologous recombination, or with recombinant productionmethods utilizing control elements introduced into cells alreadycontaining DNA encoding HRG currently in use in the field. For example,a powerful promoter/enhancer element, a suppresser, or an exogenoustranscription modulatory element is inserted in the genome of theintended host cell in proximity and orientation sufficient to influencethe transcription of DNA encoding the desired HRG. The control elementdoes not encode HRG of this invention, but the DNA is present in thehost cell genome. One next screens for cells making HRG of thisinvention, or increased or decreased levels of expression, as desired.

[0249] F. Detecting Gene Amplification/Expression

[0250] Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77: 5201-5205 (1980)), dot blotting (DNA analysis), orin situ hybridization, using an appropriately labeled probe based on thesequences provided herein. Various labels may be employed, most commonlyradioisotopes, particularly ³²p. However, other techniques may also beemployed, such as using biotin-modified nucleotides for introductioninto a polynucleotide. The biotin then serves as the site for binding toavidin or antibodies which may be labeled with a wide variety of labels,such as radionuclides, fluorescers, enzymes, or the like. Alternatively,antibodies may be employed that can recognize specific duplexes,including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes orDNA-protein duplexes. The antibodies in turn may be labeled and theassay may be carried out where the duplex is bound to a surface, so thatupon the formation of duplex on the surface, the presence of antibodybound to the duplex can be detected.

[0251] Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. With immunohistochemical stainingtechniques, a cell sample is prepared, typically by dehydration andfixation, followed by reaction with labeled antibodies specific for thegene product coupled where the labels are usually visually detectablesuch as enzymatic labels, fluorescent labels, luminescent labels, andthe like. A particularly sensitive staining technique suitable for usein the present invention is described by Hsu et al., Am. J. Clin. Path.,75: 734-738 (1980).

[0252] Antibodies useful for immunohistochemical staining and/or assayof sample fluids may be either monoclonal or polyclonal, and may beprepared in any mammal. Conveniently, the antibodies may be preparedagainst a native HRG polypeptide or against a synthetic peptide based onthe DNA sequences provided herein as described further below.

[0253] G. Purification of the Heregulin Polypeptides

[0254] HRG is recovered from a cellular membrane fraction.Alternatively, a proteolytically cleaved or a truncated expressedsoluble HRG fragment or subdomain are recovered from the culture mediumas a soluble polypeptide. A HRG is recovered from host cell lysateswhen'directly expressed without a secretory signal.

[0255] When HRG is expressed in a recombinant cell other than one ofhuman origin, HRG is completely free of proteins or polypeptides ofhuman origin. However, it is desirable to purify HRG from recombinantcell proteins or polypeptides to obtain preparations that aresubstantially homogeneous as to HRG. As a first step, the culture mediumor lysate is centrifuged to remove particulate cell debris. The membraneand soluble protein fractions are then separated. HRG is then bepurified from both the soluble protein fraction (requiring the presenceof a protease) and from the membrane fraction of the culture lysate,depending on whether HRG is membrane bound. The following procedures areexemplary of suitable purification procedures: fractionation onimmunoaffinity or ion-exchange columns; ethanol precipitation; reversephase HPLC; chromatography on silica, heparin SEPHAROSE or on a cationexchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammoniumsulfate precipitation; and gel filtration using, for example, SEPHADEXG-75.

[0256] HRG variants in which residues have been deleted, inserted orsubstituted are recovered in the same fashion as the native HRG, takingaccount of any substantial changes in properties occasioned by thevariation. For example, preparation of a HRG fusion with another proteinor polypeptide, e.g., a bacterial or viral antigen, facilitatespurification; an immunoaffinity column containing antibody to theantigen can be used to adsorb the fusion. Immunoaffinity columns such asa rabbit polyclonal anti-HRG column can be employed to absorb HRGvariant by binding it to at least one remaining immune epitope. Aprotease inhibitor such as phenylmethylsulfonylfluoride (PMSF) also maybe useful to inhibit proteolytic degradation during purification, andantibiotics may be included to prevent the growth of adventitiouscontaminants. One skilled in the art will appreciate that purificationmethods suitable for native HRG may require modification to account forchanges in the character of HRG variants or upon expression inrecombinant cell culture.

[0257] H. Covalent Modifications of HRG

[0258] Covalent modifications of HRG polypeptides are included withinthe scope of this invention. Both native HRG and amino acid sequencevariants of HRG optionally are covalently modified. One type of covalentmodification included within the scope of this invention is a HRGpolypeptide fragment. HRG fragments, such as HRG-GDF, having up to about40 amino acid residues are conveniently prepared by chemical synthesis,or by enzymatic or chemical cleavage of the full-length HRG polypeptideor HRG variant polypeptide. Other types of covalent modifications of HRGor fragments thereof are introduced into the molecule by reactingtargeted amino acid residues of HRG or fragments thereof with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

[0259] Cysteinyl residues most commonly are reacted with α-haloacetates(and corresponding amines), such as chloroacetic acid orchloroacetamide, to give carboxymethyl or carboxyamidomethylderivatives. Cysteinyl residues also are derivatized by reaction withbromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0260] Histidyl residues are derivatized by reaction withdiethylpyrocarbonate at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain. Para-bromophenacyl bromide also isuseful; the reaction is preferably performed in 0.1M sodium cacodylateat pH 6.0.

[0261] Lysinyl and amino terminal residues are reacted with succinic orother carboxylic acid anhydrides. Derivatization with these agents hasthe effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing a-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

[0262] Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

[0263] The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

[0264] Carboxyl side groups (aspartyl or glutamyl) are selectivelymodified by reaction with carbodiimides (R′—N═C═N—R′), where R and R′are different alkyl groups, such as1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

[0265] Derivatization with bifunctional agents is useful forcrosslinking HRG to a water-insoluble support matrix or surface for usein a method for purifying anti-HRG antibodies, and vice versa. Commonlyused crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacids homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-((p-azidophenyl)dithio)propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

[0266] Glutaminyl and asparaginyl residues are frequently deamidated tothe corresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

[0267] Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the a-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

[0268] HRG optionally is fused with a polypeptide heterologous to HRG.The heterologous polypeptide optionally is an anchor sequence such asthat found in a phage coat protein such as M13 gene IlIl or gene VIIIproteins. These heterologous potypeptides can be covalently coupled toHRG polypeptide through side chains or through the terminal residues.

[0269] HRG may also be covalently modified by altering its nativeglycosylation pattern. One or more carbohydrate substituents in theseembodiments, are modified by adding, removing or varying themonosaccharide components at a given site, or by modifying residues inHRG as that glycosylation sites are added or deleted.

[0270] Glycosylation of polypeptides is typically either N-linked orO-linked. N-linked refers to the attachment of the carbohydrate moietyto the side chain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

[0271] Glycosylation sites are added to HRG by altering its amino acidsequence to contain one or more of the above-described tri-peptidesequences (for N-linked glycosylation sites). The alteration may also bemade by the addition of, or substitution by, one or more serine orthreonine residues to HRG (for O-linked glycosylation sites). For ease,HRG is preferably altered through changes at the DNA level, particularlyby mutating the DNA encoding HRG at preselected bases such that codonsare generated that will translate into the desired amino acids.

[0272] Chemical or enzymatic coupling of glycosides to HRG increases thenumber of carbohydrate substituents. These procedures are advantageousin that they do not require production of the polypeptide in a host cellthat is capable of N- and O-linked glycosylation. Depending on thecoupling mode used, the sugar(s) may be attached to (a) arginine andhistidine, (b) free carboxyl groups, (c) free sulfhydryl groups such asthose of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330, published Sep.11, 1987, and in Aplin and Wriston (CRC Crit. Rev. Biochem., pp. 259-306(1981)).

[0273] Carbohydrate moieties present on an HRG also are removedchemically or enzymatically. Chemical deglycosylation requires exposureof the polypeptide to the compound trifluoromethanesulfonic acid, or anequivalent compound. This treatment results in the cleavage of most orall sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the polypeptide intact. Chemicaldeglycosylation is described by Hakimuddin et aL (Arch. Biochem.Biophys., 259:52 (1987)) and by Edge et al. (Anal. Biochem., 118:131(1981)). Carbohydrate moieties are removed from HRG by a variety ofendo- and exo-glycosidases as described by Thotakura et al. (Meth.Enzymol., 138:350 (1987)).

[0274] Glycosylation also is suppressed by tunicamycin as described byDuskin et al. (J. Biol Chem., 257:3105 (1982)). Tunicamycin blocks theformation of protein-N-glycoside linkages.

[0275] HRG may also be modified by linking HRG to variousnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

[0276] One preferred way to increase the in vivo circulating half lifeof non-membrane bound HRG is to conjugate it to a polymer that confersextended half-life, such as polyethylene glycol (PEG). (Maxfield, et al,Polymer 16,505-509 (1975); Bailey, F. E., et al, in Nonionic Surfactants(Schick, M. J., ed.) pp.794-821, 1967); (Abuchowski, A. et al., J. Biol.Chem. 252, 3582-3586, 1977; Abuchowski, A. et al., Cancer Biochem.Biophys. 7, 175-186, 1984); (Katre, N. V. et al., Proc. Natl. Acad.Sci., 84, 1487-1491, 1987; Goodson, R. et al. Bio Technology, 8,343-346, 1990). Conjugation to PEG also has been reported to havereduced immunogenicity and toxicity (Abuchowski, A. et al., J. Biol.Chem., 252, 3578-3581, 1977).

[0277] HRG may also be entrapped in microcapsules prepared, for example,by coacervation techniques or by interfacial polymerization (forexample, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Osol, A., Ed., (1980).

[0278] Those skilled in the art will be capable of screening variants inorder to select the optimal variant for the purpose intended. Forexample, a change in the immunological character of HRG, such as achange in affinity for a given antigen or for the HER2 receptor, ismeasured by a competitive-type immunoassay using a standard or controlsuch as a native HRG (in particular native HRG-GFD). Other potentialmodifications of protein or polypeptide properties such as redox orthermal stability, hydrophobicity, susceptibility to proteolyticdegradation, stability in recombinant cell culture or in plasma, or thetendency to aggregate with carriers or into multimers are assayed bymethods well known in the art.

[0279] I. Heregulin Antibody Preparation

[0280] The antibodies of this invention are obtained by routinescreening and include polyclonal antibodies, monoclonal antibodies andfragments thereof.

[0281] Polyclonal antibodies are preferably raised in animals bymultiple subcutaneous (sc) or intraperitoneal (ip) injections of therelevant antigen and an adjuvant. It may be useful to conjugate therelevant antigen to a protein that is immunogenic in the species to beimmunized, e.g., keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor using a bifunctional orderivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, SOCl₂, orR¹N═C═NR, where R and R¹ are different alkyl groups.

[0282] Animals are immunized against the antigen, immunogenicconjugates, or derivatives by combining, e.g., 100 μg or 5 μg of theprotein or conjugate (for rabbits or mice, respectively) with 3 volumesof Freund's complete adjuvant and injecting the solution intradermallyat multiple sites. One month later the animals are boosted with ⅕ to{fraction (1/10)} the original amount of peptide or conjugate inFreund's complete adjuvant by subcutaneous injection at multiple sites.Seven to 14 days later the animals are bled and the serum is assayed forantibody titer. Animals are boosted until the titer plateaus.Preferably, the animal is boosted with the conjugate of the sameantigen, but conjugated to a different protein and/or through adifferent cross-linking reagent. Conjugates also can be made inrecombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are suitably used to enhance the immune response.

[0283] Monoclonal antibodies may be made using the hybridoma methodfirst described by Kohler et al., Nature, 256:495 (1975), or may be madeby recombinant DNA methods (U.S. Pat. No. 4,816,567).

[0284] In the hybridoma method, a mouse or other appropriate hostanimal, such as a hamster or macaque monkey, is immunized as hereinabovedescribed to elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the protein used forimmunization. Alternatively, lymphocytes may be immunized in vitro.Lymphocytes then are fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principles and Practice, pp.59-103 (AcademicPress, 1986)).

[0285] The hybridoma cells thus prepared are seeded and grown in asuitable culture medium that preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells. For example, if the parental myeloma cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (HAT medium), which substances prevent thegrowth of HGPRT-deficient cells.

[0286] Preferred myeloma cells are those that fuse efficiently, supportstable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these, preferred myeloma cell lines are murine myelomalines, such as those derived from MOP-21 and M.C.-11 mouse tumorsavailable from the Salk Institute Cell Distribution Center, San Diego,Calif. USA, and SP-2 or X63-Ag8-653 cells available from the AmericanType Culture Collection, Rockville, Md. USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0287] Culture medium in which hybridoma cells are growing is assayedfor production of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

[0288] The binding affinity of the monoclonal antibody can, for example,be determined by the Scatchard analysis of Munson et al., Anal.Biochem., 107:220 (1980).

[0289] After hybridoma cells are identified that produce antibodies ofthe desired specifcity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

[0290] The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-SEPHAROSE, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0291] DNA encoding the monoclonal antibodies is readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of the monoclonal antibodies). The hybridomacells serve as a preferred source of such DNA. Once isolated, the DNAmay be placed into expression vectors, which are then transfected intohost cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. Recombinant production of antibodies willbe described in more detail below.

[0292] Hybridoma cell lines producing antibodies are identified byscreening the culture supernatants for antibody which binds to HER2/HER3receptors. This is routinely accomplished by conventional immunoassaysusing soluble receptor preparations or by FACS using cell-bound receptorand labeled candidate antibody. Agonist antibodies are preferablyantibodies which stimulate autophosphorylation in the HRG tyrosineautophosphorylation assay described above.

[0293] The hybrid cell lines can be maintained in culture in vitro incell culture media. The cell lines of this invention can be selectedand/or maintained in a composition comprising the continuous cell linein hypoxanthine-aminopterin thymidine (HAT) medium. In fact, once thehybridoma cell line is established, it can be maintained on a variety ofnutritionally adequate media. Moreover, the hybrid cell lines can bestored and preserved in any number of conventional ways, includingfreezing and storage under liquid nitrogen. Frozen cell lines can berevived and cultured indefinitely with resumed synthesis and secretionof monoclonal antibody. The secreted antibody is recovered from tissueculture supernatant by conventional methods such as precipitation, ionexchange chromatography, affinity chromatography, or the like. Theantibodies described herein are also recovered from hybridoma cellcultures by conventional methods for purification of IgG or IgM as thecase may be that heretofore have been used to purify theseimmunoglobulins from pooled plasma, e.g., ethanol or polyethylene glycolprecipitation procedures.

[0294] Human antibodies may be used and are preferable. Such antibodiescan be obtained by using human hybridomas (Cote et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985)). Chimericantibodies, Cabilly et al., U.S. Pat. No. 4,816,567, (Morrison et al.,Proc. Natl. Acad. Sci., 81:6851 (1984); Neuberger et al., Nature 312:604(1984); Takeda et al., Nature 314:452 (1985)) containing a murineanti-HER21HER3 variable region and a human constant region ofappropriate biological activity (such as ability to activate humancomplement and mediate ADCC) are within the scope of this invention, asare humanized antibodies produced by conventional CDR-grafting methods(Riechmann et al., Nature 332:333-327(1988EP 0328404 A1; EP 02394000A2).

[0295] Techniques for creating recombinant DNA versions of theantigen-binding regions of antibody molecules (Fab or variable regionsfragments) which bypass the generation of monoclonal antibodies are alsoencompassed within the practice of this invention. One extractsantibody-specific messenger RNA molecules from immune system cells takenfrom an immunized subject, transcribes these into complementary DNA(cDNA), and clones the cDNA into a bacterial expression system andselects for the desired binding characteristic. TheScripps/Stratagenemethod uses a bacteriophage lambda vector systemcontaining a leader sequence that causes the expressed Fab protein tomigrate to the periplasmic space (between the bacterial cell membraneand the cell wall) or to be secreted. One can rapidly generate andscreen great numbers of functional Fab fragments to identify those whichbind the receptors with the desired characteristics. Alternatively, theantibodies can be prepared by the phage display techniques described inHoogenboom, Tibtech February 1997 (vol 15); Neri et al., Cell Biophysics27:47-61 (1995); Winter et al., Annu. Rev. Immunol. 12:433-55 (1994);and Soderlind et al., Immunol. Rev. 130:109-124 (1992) and thereferences described therein as well as the monovalent phage displaytechnique described in Lowman et al., Biochem. 30:10832-10838 (1991).

[0296] 2. Therapeutic compositions, administration and use of Heregulinsand Agonist Antibodies

[0297] The HRG are used in the present invention to induce epithelialcell growth, for example lung epithelial cell growth, proliferation anddifferentiation, and to increase the production of surfactant protein Aby lung cells. These effects allow treatment of disease statesassociated with tissue damage, for example, chronic obstructivepulmonary disease(COPD) including subtypes thereof such as chronicbronchitis, emphysema, asthma, etc., neonatal pulmonary diseasesincluding neonatal respiratory distress syndrome, meconium aspirationsyndrome, chronic lung disease of the neonate, congenital diaphragmatichernia, etc., acute lung injuries including smoke or chemicalinhalation, pneumonitis due to aspiration, radiation, etc., neardrowning, cystic fibrosis and other epithelial cell trauma diseases,including injuries associated with surgical wounds and resections,ulcers, lesions, and tissue tears, with the method of the invention.

[0298] A preferred indication for treatment with the method of theinvention is the treatment of COPD. COPD is a spectrum of chronicinflammatory respiratory diseases characterized by cough, sputum,dyspnea, airflow limitation and impaired gas exchange. COPD is common inolder populations and presents a pattern of gradually declining lungfunction. Typically, a patient will exhibit a chronic cough with clearsputum which worsens to a cough with thick sputum and accompanying poorair exchange. These conditions frequently lead to heart disease anddeath. Many persons with COPD will have chronic bronchitis together withemphysema. The present invention is particularly important because ithalts, slows and/or reverses the lung destruction process in COPDpatients. In this action, the method of the invention is very differentfrom typical treatments for COPD in which the symptoms are treated, butnot the underlying destruction of lung cell tissue and function.

[0299] The method of the invention may, however, be combined with oradministered together with other therapies for treatment of lung diseasesuch as COPD. For example, the method of this invention can be usedtogether with the administration of an anticholinergic bronchodilatorsuch as ipratropium bromide (ATROVENT available from BoehringerIngleheim) or tiotropium, a beta adrenergic receptor agonist such asalbuterol (PROVENTIL available from Schering) or salmeterol, steroidssuch as prednisone, retinoic acid, phosphodiesterase inhibitors,endothelin antagonists, metalloproteinase inhibitors, elastaseinhibitors, free radical inhibitors, serine proteinase inhibitors,neutrophil elastase inhibitors, pulmonary surfactant compositions suchas beractant (SURVANTA available from Ross Labs.), PDGF, FGF, EGF,growth hormone or other protein growth factors, etc. or combinationsthereof. The relative amount of the HRG and the additional compound(s)can be readily determined by a physician with regard to the individualsymptoms of the patient. It is anticipated that these compositions andthe relative amounts of the components therein will be varied asnecessary to address the specific needs of a patient and will bemonitored and adjusted using conventional physiochemical and medicaltests for lung function.

[0300] Therapeutic formulations of HRG or agonist antibody are preparedfor storage by mixing the HRG protein having the desired degree ofpurity with optional physiologically acceptable carriers, excipients, orstabilizers (Remington's Pharmaceutical Sciences, supra), in the form oflyophilized cake or aqueous solutions. Acceptable carriers, excipientsor stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid; lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN, PLURONICS or polyethylene glycol (PEG).

[0301] HRG or agonist antibody to be used for in vivo administrationmust be sterile. This is readily accomplished by filtration throughsterile filtration membranes, prior to or following lyophilization andreconstitution. The HRG or antibody ordinarily will be stored inlyophilized form or in solution.

[0302] Therapeutic HRG or antibody compositions generally are placedinto a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

[0303] The route of HRG or antibody administration is in accord withknown methods, e.g., injection or infusion by intravenous,intraperitoneal, intracerebral, intramuscular, intraocular,intraarterial, powder or liquid aerosol administration to the nose orlung or intralesional routes, or by sustained release systems as notedbelow. The HRG ligand may be administered continuously by infusion or bybolus injection. An agonist antibody is preferably administered in thesame fashion, or by administration into the blood stream or lymph.

[0304] The HRG, HRG variant or fragment and agonist antibodies may bespray dried or spray freeze dried using known techniques (Yeo et al,Biotech. and Bioeng., 41:341-346 (1993); Gombotz et al, PCT/US90/02421).

[0305] Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theprotein, which matrices are in the form of shaped articles, e.g. films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981)and Langer, Chem. Tech., 12: 98-105 (1982) or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22: 547-556 (1983)), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLUPRON DEPOT (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid (EP 133,988). While polymers such as ethylene-vinyl acetate andlactic acid-glycolic acid enable release of molecules for over 100 days,certain hydrogels release proteins for shorter time periods. Whenencapsulated proteins remain in the body for a long time, they maydenature or aggregate as a result of exposure to moisture at 37° C.,resulting in a loss of biological activity and possible changes inimmunogenicity. Rational strategies can be devised for proteinstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

[0306] Sustained-release HRG or antibody compositions also includeliposomally entrapped HRG or antibody. Liposomes containing HRG orantibody are prepared by methods known per se: DE 3,218,121; Epstein etal., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676;EP 88,046; EP 143,949; EP 142,641; Japanese patent application83-118008; U.S. Pats. No. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily the liposomes are of the small (about 200-800 Angstroms)unilamelar type in which the lipid content is greater than about 30 mol.% cholesterol, the selected proportion being adjusted for the optimalHRG therapy. Liposomes with enhanced circulation time are disclosed inU.S. Pat. No. 5,013,556.

[0307] An effective amount of HRG or antibody to be employedtherapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, and the condition of thepatient. Also, the amount of HRG polypeptide will generally be less thanthe amount of an agonist antibody. Accordingly, it will be necessary forthe therapist to titer the dosage and modify the route of administrationas required to obtain the optimal therapeutic effect. A typical dailydosage might range from about 1 μg/kg to about 1 mg/kg and up to 100mg/kg or more, depending on the factors mentioned above. Typically, theclinician will administer HRG or antibody until a dosage is reached thatachieves the desired effect. The progress of this therapy is easilymonitored by conventional assays, for example, surfactant protein Aproduction.

[0308] In a further embodiment, epithelial cells may be obtained orisolated from a mammalian tissue to obtain a normal epithelial cellsample using techniques well known in the art (biopsy, etc.). Thissample may then be treated with a heregulin protein in order to induceepithelial cell growth and/or proliferation in the sample therebyexpanding the population of primary epithelial cells. Typically,heregulin will be added to the in vitro epithelial cell culture at aconcentration of about 0.1 to about 100 nM preferably 1-50 nM. Ifdesired, the primary epithelial cells may be cultured in vitro forseveral generations in order to sufficiently expand the epithelial cellpopulation. The epithelial cells are cultured under conditions suitablefor mammalian cell culture as discussed above. After expansion, theexpanded sample is reintroduced into the mammal for the purpose ofre-epithelializing the mammalian tissue. For example, lung epithelialcells isolated from a patient having emphysema or chronic obstructivepulmonary disease may be obtained, expanded and reintroduced into thelung in order to more quickly re-epithelialize the damaged lung tissuethereby reestablishing lung function. The expanded cells may bereintroduced into the lung by aspiration or intubafion using methodswell known in the art.

[0309] The methods and procedures described herein with respect to HRG-αor HRG in general may be applied similarly to other HRG such as HRG-β1,HRG-β2 and HRG-β3 and to variants thereof, as well as to the antibodies.All references cited in this specification are expressly incorporated byreference. The following examples are offered by way of illustration andnot by way of limitation.

EXAMPLES Example 1

[0310] Preparation of Heregulins

[0311] (a) Heregulins HRG-α, HRG-β1, HRG-β2, HRG-β2-like, and HRG-β3were isolated, cloned, expressed and isolated from the cell culturemedium as described in U.S. Pat. No. 5,367,060.

[0312] (b) SMDF polypeptides are prepared as described in WO 96/15244.

[0313] (c) γ-HRG polypeptide was prepared and characterized as describedbelow.

[0314] Reagents:

[0315] The EGF-like domain of HRGβ1₍₁₇₇₋₂₄₄₎ was expressed in E coli,purified and radioiodinated as described previously (Sliwkowski et al.J. Biol. Chem. 269:14661-14665 (1994)). The anti-HER2 monoclonalantibodies 2C4 and 4D5 have been described elsewhere (Fendly et al.Cancer Research 50:1550-1558 (1990)).

[0316] HER3 and HER4-immunoadhesins:

[0317] A unique Ml I site was engineered into a plasmid expressing humanIgG heavy chain at the region encoding the hinge domain of theimmunoglobulin. Ml I sites were also engineered into a set of HERexpression plasmids at the region encoding the ECD/TM junctions of thesereceptors. All mutageneses were done using the Kunkel method (Kunkel,T., Proc. Natl. Acad. Sci. U.S.A. 82:488 (1985)). The Ml I sites wereutilized to make the appropriate HER-IgG fusion constructs. The fusionjunctions of the various HER-IgG chimeras were: for HER2, E⁶⁴⁶_(HER2)-(TR)-DKTH²²⁴ _(VH); for HER3, L⁶³⁶ _(HER3)-(TR)-DKTH²²⁴ _(VH);for HER4, G⁶⁴⁰ _(HER4)-(TR)-DKTH²²⁴ _(VH). The conserved TR sequence isderived from the Ml I site. The final expression constructs were in apRK-type plasmid backbone wherein eukaryotic expression is driven by aCMV promoter (Gorman et al., DNA Prot. Eng. Tech. 2:3-10 (1990)).

[0318] To obtain protein for in vitro experiments, adherent HEK-293cells were transfected with the appropriate expression plasmids usingstandard calcium phosphate methods (Gorman et al., supra and Huang etal., Nucleic Acids Res. 18:937-947 (1990)). Serum-containing media wasreplaced with serum-free media 15 hours post-transfection and thetransfected cells incubated for 5-7 days. The resulting conditionedmedia was harvested and passed through Protein A columns (1 mL PharmaciaHiTrap™). Purified IgG fusions were eluted with 0.1 M citric acid (pH4.2) into tubes containing 1 M Tris pH 9.0. The eluted proteins weresubsequently dialyzed against PBS and concentrated using Centri-prep-30filters (Amicon). Glycerol was added to a final concentration of 25% andthe material stored at -20° C. Concentrations of material weredetermined via a Fc-ELISA. Cell Culture: Human breast cancer cell linesMDA-MB-175, MDA-MB-231, SK-BR-3 and MCF7 were obtained from the AmericanType Culture Collection and maintained in a 50:50 mixture of F12 Ham'sand Dulbecco's modified Eagle medium (DMEM), supplemented with 10% heatinactivated FBS, 2 mM glutamine and 10% penicillin-streptomycin.

[0319] Generation and Characterization of cDNA Library:

[0320] Total RNA was purified from MDA-MB-175 cells using theguanidinium isothiocyanate-cesium chloride procedure (Sambrook et al.,Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, (1989)). Poly (A)⁺ RNA was isolated using oligo (dT)Dynabeads (DYNAL) as recommended by the supplier. First and secondstrand syntheses were performed using a Gibco BRL cDNA synthesis kit.λgt10 cDNA recombinants were generated when a cDNA cloning system fromAmersham was used. In vitro packaging was performed using Gigapack IIIpackaging extract (Stratagene). PstI-Xhol HRG03 cDNA fragment (nt144-618) was labeled by random priming and 1×10⁶ plaques were screened.Positive clones were confirmed and purified by secondary and tertiaryscreening. Phage DNA was isolated as a BamHI fragment and subcloned intothe corresponding site of pBluescript SK⁻. Clone 5 was completelysequenced using the Sequenase version 2.0

DNA sequencing kit (United States Biochemicals, Inc.). Both strands weresequenced.

[0321] Bacterial Expression System:

[0322] A cDNA fragment of clone 5 (nt 1690-2722) was subcdoned into thepET-32 TRX fusion vector (Novagen). This BgIII-BgIII fragment wasinserted into the BamHI site of the pET32a plasmid. The trxy-HRG (aminoacids 455-768) protein expression in E. coli was induced as recommendedby the supplier.

[0323] Purification of Recombinant γ-HRG:

[0324]E. coli cells expressing trxγ-HRG were collected and suspended at9 ml/g in 50 mM Tris HCL pH 8. Lysozyme was added to a finalconcentration of 0.2 mg/ml and the solution was stirred on ice for 1 hr.Dnase I (10 μg/ml) and MgCl₂ (4 mM) were added. The solution was thensonicated for 30 min and cell pellets collected afterwards. The pelletfraction was dissolved at 250 ml/g in 6 M Gdn HCL, 0.1 M Tris HCL, pH8.8. Solubilized proteins were sulfitolyzed by adding 1/10 volume of 1 MNa₂SO₃ and 1110 volume of 0.2 M Na₂S₄0₆. The reaction was allowed toproceed for 1.5 hours at room temperature and protein was purified bygel filtration chromatography using a High Load Superdex™ 75 prep gradecolumn (Pharmacia). Refolding was initiated by the addition of 1 mMcysteine, and 10 mM methionine was added as an antioxidant and incubatedovernight at room temperature. Protein concentration was determined byquantitative amino acid analysis.

[0325] Northern and Southern Hybridization:

[0326] Total RNA was isolated by the method of Chomczynski et al. AnalBiochem. 162:156-159 (1987). Poly (A)⁺ was isolated using oligo d(T)cellulose columns (Qiagen) as recommended by the supplier. RNA wasdenatured and size fractioned in a 0.8% formaldehyde/1% agarose gel andtransferred onto nylon membrane (Hybond, Amersham). RNA was UVcrosslinked (UV Stratalinker, Stratagene). Prehybridization was carriedout at 42° C. in 50% formamide/1% SDS/1 M NaCl, 10% dextransulfate and100 μg/ml herring sperm DNA for at least 2 hours. cDNA probes usingeither a restriction fragment with complementary sequence to theEGF-like domain of HRGP3 or a KpnI-AvaII cDNA fragment encoding theunique sequence of γ-HRG (nt 1238-1868) were radiolabeled by randompriming (Prime-It II, Stratagene). Hybridiziation was done in equalsolution at 42° C. containing the ³²p labeled fragments for 16 hr. Blotswere washed several times with 2×SSC/1% SDS at room temperature, washedwith the same solution at 65° C. for 20 min and finally washed with0.2×SSC/0.1% SDS at room temperature for 15 min. The blots were airdried and exposed to Du Pont Reflection™ film with intensifying screensat −80° C. for 7-40 hours. Human multiple tissue Northern blots(Clontech) containing 2 μg poly (A)⁺ from spleen, thymus, prostate,testis, ovary, small intestine, colon, peripheral blood leukocytes,heart, brain, placenta, lung, liver, skeletal muscle, kidney andpancreas were hybridized with a radiolabeled γ-HRG cDNA probe (nt841-1447) as recommended by the supplier.

[0327] MDA-MB-175 and MDA-MB-231 genomic DNA was isolated as describedin Sambrook et al, supra. DNA was digested with different restrictionenzymes, prior to transfer treated with 0.25 N HCl and transferred ontonylon membrane (Hybond, Amersham). Bglll-Ndel cDNA fragment of γ-HRG (nt1690-2351) was also radiolabeled by random priming and used as ahybridization probe. Prehybridization was carried out in6×SSC/5×Denhardt's/0.75% SDS, 10% Dextransulfate and 100 μg/ml herringsperm DNA at 68° C. for 4 hours and hybridization with radiolabeledprobe was done overnight. The same wash conditions as for Northern blotswere used except a wash step with 0.2×SSC/0.1% SDS at 68° C. was addedand detection was pursued as described above.

[0328]¹²⁵I-HRG Binding Assay:

[0329] Binding assays were performed in Nunc breakapart strip wells.Plates were coated at 4° C. overnight with 100 μl of 5 μg/mlgoat-anti-human antibody (Boehringer Mannheim) in 50 mM carbonate buffer(pH 9.6). Plates were rinsed twice with wash buffer (PBS/0.05%Tween-200) and blocked with 100 μl 1% BSA/PBS for 30 min. Buffer wasremoved and each well was incubated with 15 ng IgG fusion protein in 1%BSA/PBS under vigorous shaking for 1.5 hours. Plates were rinsed threetimes with wash buffer and competitive binding was carried out by addingvarious amounts of γ-HRG and ¹²⁵I-HRGβ1 under vigorous shaking. Afterincubation for 1.5-2 hours, wells were rinsed three times with washbuffer, drained and individual wells were counted using a 100 Series IsoData γ-counter.

[0330] Tyrosine Phosphorylation Assay:

[0331] MCF7 cells were plated in 24 well plates at 1×10⁵ cells/well inF12/DMEM containing 10% FBS. After 48 hours, cells were washed withserum free F12/DMEM and serum starved for 6 hours. Variousconcentrations of bacterial expressed truncated γ-HRG (i.e., 0 pM, 22pM, 66 pM, 200 pM and 600 pM trxγ-HRG) or unpurified conditioned mediumof MDA-MB-175 cells were prepared in binding buffer (0.1% BSA inF12/DMEM) and added to each well. After 8 min incubation at roomtemperature, media was carefully aspirated and reactions were stopped byadding 100 μl of sample buffer (5% SDS, 0.25% 2-mercaptoethanol, 25 mMTris-HCL pH 6-8). 20 μl of each sample was size fractionated in a 4-12%gradient gel (Novex) and then electrophoretically transferred ontonitrocellulose membrane. Antiphosphotyrosine (4G10, from UBI, used at 1μg/ml) immunoblots were developed and the predominant reactive band atM_(r)˜180 kDa was quantified by reflectance densitometry.

[0332] Production and Characterization of Conditioned Medium fromMDA-MB-175 Cells.

[0333] Cells were seeded in T175 flasks and grown until reaching 70-80%confluency (˜2.5×10⁷ cells/flask). Subsequently, cells were washed withPBS and grown in serum free F12/DMEM medium for 3-4 days. Medium wasthen collected, filtered and concentrated using an ultrafiltration cellwith YM10 Diaflo ultafiltration membranes (Amicon). γ-HRG was visualizedin conditioned medium of MDA-MB-175 cells by Western blot analysis undernon reducing conditions. γ-HRG was partially purified by HPLC using a C4reverse phase column. CHO expressed full length HRGp1 (lane 1) and semipure γ-HRG (lane 2) were electrophoresed, blot was probed with HER2/HER4IgG heterodimers and Western blot was developed. A ˜64 kDa band could beseen in the lane containing partial purified supernatant whereas CHOexpressed full length HRGβ1 migrated as a 45 kDa protein.

[0334] Cell Proliferation Assay with Crystal Violet:

[0335] Tumor cell lines were plated in 96 well plates at followingdensities: 2×10⁴ cells/well for MDA-MB-175 and 1×10⁴ cells/well forSK-BR-3. The media contained 1% FBS and cells were allowed to adhere for2 hours. Monoclonal antibodies, immunoadhesions (10 μg/ml) or mediaalone were added and the cells were incubated for 2 hours at 37° C.rHRGβ177-244 was added at a final concentration of 1 nM, or 100 nM forneutralising the immunoadhesion, and the cells were incubated for 4days. Monolayers were washed with PBS and stained/fixed with 0.5%crystal violet. Plates were air dried, the dye was eluted with 0.1 Msodium citrate (pH 4.2) in ethanol (50:50) and the absorbance wasmeasured at 540 nm.

[0336] Isolation and Sequence Analysis of γ-HRG:

[0337] To characterize the heregulin transcript in MDA-MB-175 cells, aλgt 10 cDNA library was constructed with mRNA derived from this cellline. The library was screened with a cDNA probe corresponding to theEGF-like domain and part of the N-terminal sequence of HRGP3. Variousclones were identified. One of the clones which appeared to contain thefull length CDNA sequence was isolated and sequenced. FIGS. 7A-7D showsthe nucleotide sequence and the predicted amino acid sequence of y-HRG.The single open reading frame of 2303 bp starts with an ATG codon at nt334. This start codon lies in a nucleotide sequence context, which isknown to be a potential translation initiation site (Kozak, Nucleic AcidResearch 15:8125-8148 (1987)). Several termination codons were foundupstream of the initiation codon. The stop codon TAG at nt 2637 isfollowed by the 3′ noncoding sequence, which is identical to other HRGisoform sequences and includes a polyadenylation signal followed by anA-rich region. The open reading frame encodes a protein of 768 aminoacid residues with a calculated molecular mass of 84.2 kDa.

[0338] (d) The selection of HRG-β1 variants containing residuescorresponding to the minimal EGF-like domain (HRG-β1 177-228) wasconducted using monovalent phage display. For these variants, residuenumbers also are expressed, in parentheses, in terms of the position ofthe residue in the minimal EGF-like domain (i.e., HRG-β1 EGF 1-52).

[0339] Variants of HRG-β1 EGF were prepared and selected for binding toHER-3-Ig using monovalent phage display, according to the method of Basset al., Proteins 8:309-314 (1990). As discussed in detail below, anHRG-β1 EGF phagemid vector was prepared, in which HRG-β1 EGF was fusedto a C-terminal fragment of the M13 coat protein pIII. Kunkelmutagenesis was performed to introduce stop codons into this vector atsites selected for randomization. This step ensures that the startingvector is incapable of expressing the wild-type polypeptide. Stretchesof four to six residues per library were randomized in a linear fashion,except for the six cysteines, Phel89 (HRG-β1 EGF Phel3) and the two mostC-terminal residues. Phe189 was not altered because this residue isconserved as an aromatic residue in EGF and TGF-α and forms a stackinginteraction with Tyr208 (HRG-β1 EGF Tyr32) Jacobsen et al., Biochemistry35:3402-17 (1996). HRG-β1 EGF was thus covered in eight libraries,designated A-E, G, H and I.

[0340] Library E, covering HRG-β1 202-209 (HRG-β1 EGF 26-33), containeda three-residue deletion. The deleted region corresponds to a disorderedturn between the second and third β-sheet of HRG-β1 EGF, and theequivalent amino acids are absent in EGF and TGF-α. An HRG-β1 EGFcontrol variant in which HRG-β1 202-204 (HRG-β1 EGF 26-28) of HRG8 aredeleted (HRG63) bound HER-3-Ig with an affinity similar to that ofwild-type.

[0341] An additional library (F) was created to randomize a surfacepatch composed of side chains from the first and second β-sheets, whichincluded HRG-β1 178, 180, 198, and 200 (HRG-β1 EGF 2, 4, 22, and 24).

[0342] The selected sites in the starting vectors were randomized byKunkel mutagenesis to produce HRG-β1 EGF libraries. Phage displayingmutated HRG-β1 EGFs were produced from the libraries under conditionssuch that, statistically, each phage particle displayed no more than onecopy of the mutated HRG-β1 EGF. See Bass et al., supra. These phage werethen selected for binding to (sorted against) HER-3-Ig immobilized on anELISA plate. Bound phage were eluted and used to reinfect host cells,which were used to produce new phage for another round of sorting. Thisprocess was repeated six to seven times for each library. Twelve clonesfrom the phage selected from each library were then sequenced. TABLE 2Library A Variants Position in HRG-β1 Variant No. 177 178 179 180 181Wild-type S H L V K  1 W R . . P  2 W S . Q P  3, 5, 10 W E . . P  4 W S. . .  6 W S . I P  7 W R . . A  8 W A . . P  9 W S . Q . 11 W E . . A12 W S . E P

[0343] TABLE 3 Library B Variants Position in HRG-β1 Variant No. 183 184185 186 187 188 Wild-type A E K E K T  1* G V G R D G  2* G G E R E G  3G . E R E G  4*, 5* G W D R E G  6* G V Q R E G  7 G . E R A G  8 G K ER E G  9* T N S R E G 10* D K S R E G 11* G . D R . Q 12 G R E R E G

[0344] TABLE 4 Library C Variants Position in HRG-β1 Variant No. 191 192193 194 195 Wild-type V N G G E 1, 2, 4, 5, 7-12 . . . . . 3 . . . . V 6. . . . Q

[0345] TABLE 5 Library D Variants Position in HRG-β1 Variant No. 197 198199 200 201 Wild-type F M V K D  1*, 2*, 8*, 12* Y K . R I  3 . R . . T 4, 5, 7, 9 Y R . . T  6 Y . I . Y 10 Y . . . T 11 M R . R T

[0346] TABLE 6 Library E Variants Position in HRG-β1 Variant No. 205 206207 208 209 Wild-type P S R Y L  1 T P Y L M  2, 4 Y G Y L M  3* Y R Y RM  5, 12 T H Y R G  6 T H Y R M  7* Y K Y R M  8, 9 T K Y R G 10 Y K Y R.

[0347] TABLE 7 Library G Variants Position in HRG-β1 Variant No. 211 212213 214 215 216 Wild-type K C P N E F  1, 5, 6, 10, 12 R . S L . .  2 R. S E . .  3 . . . K . M  4 R . T V . Y  7, 8 R . T V . Y  9 . . N S . .11 R . K K . .

Example 2

[0348] HER2/HER3 Expression in Embryonic Rat Lung

[0349] Rat lungs were microdissected from rat embryos on embryologic day(E) 16, 18, 20, and post-natal (P) days 7, 14, and adult. Isolated lungtissue was homogenized in a standard protease inhibitor buffer, andequal protein amounts subjected to SDS-PAGE (4-20%), blotted tonitrocellulose and identified with specific antibodies (HER2, HER3,HER4-Santa Cruz Biological, San Jose, Calif. and HRG, 3G11, Genentech,Inc.) using chemiluminescent techniques.

[0350] Analysis of the blot indicates that HER2 is expressed at highlevels throughout development in utero, appears to peak on E18 anddeclines after birth to lower adult levels. HER3 expression peaks atE18, and then declines after birth to lower adult levels. HER4 is notidentified at any time during lung development. A proform of HRG may bepresent (75 kDa protein) on E16, peaking on E18 and then declining tolow levels in the adult. These data suggest that the HER/HRG system isdevelopmentally modulated during lung development and the activereceptor may be a HER2/HER3 heterodimer. The time period during whichthe receptors peak in expression (E18) represents the pseudoglandularstage of rat lung development (days 13-18), bordering on the canalicularstage (days 19-20). During the pseudoglandular stage, pulmonaryepithelial cell proliferation is higher than at any other time. Duringthe canalicular stage, differentiation begins with the appearance oftype I pneymocyte and type II (surfactant producing) pneymocytes. Thisindicates a role for HRGIHER interaction in either or both processes.

Example 3

[0351] HER2/HER3 Expression in Fetal Human Lung

[0352] Fetal human lung was obtained from mid trimester embryos (17-22weeks). Lung tissue was cultured in serum free Weymouth's media at anair fluid interface at 37° C. in a humidified 5% CO₂ atmosphere. Tissuewas harvested from the culture on days 0 (day tissue was received) andafter 1-4 days (D) in culture (D1-D4), homogenized in a standardprotease inhibitor buffer, equal protein amounts subjected to SDS-PAGE(4-20%), blotted to nitrocellulose and identified with specificantibodies.

[0353] HER2 is expressed on DO and increases in expression level duringin vitro culture. HER3 is present at low to undetectable levels at DOand increases in expression level during in vitro culture. HER4 was notidentified at any time during lung development. HRG was not identifiedon D0, however, it was identified as a 75,000 Da protein on D1-D2 andcontinued to rise in expression level throughout time in culture. Thishuman explant model recapitulates part of the normal lung developmentalprogram over the 5 days in culture. Development occurs rapidly with bothepithelial cell proliferation, and differentiation occurring, along withair space formation. These data indicate that the HER/HRG system is alsomodulated during in vitro human lung development, and the activereceptor may be a HER2/HER3 heterodimer. The third trimester representslate pseudoglandular (days 42-112) and canalicular stage (days 112-196)in human lung development. As in the rat, during the pseudoglandularstage epithelial cell proliferation and formation of the prospectiveairways occurs. During the canalicular stage differentiation of theepithelium occurs.

Example 4

[0354] Expression of HER2 and HER3 in Human Lung

[0355] HER2 and HER3 are expressed exclusively in the pulmonaryepithelium during lung development. Mid-trimester human lung wascultured in vitro as outlined above. Tissue was harvested daily, snapfrozen, and 5 micron sections cut. The sections were mounted on glassslides, and immunohistochemistry performed using standard ABCprocedures. HER2, HER3, and HER4 were identified using specificantibodies as described above.

[0356] As a control, lung tissue was stained at D0 and D5 with anirrelevant antibody and no staining was detected. At D0, the lung isrelatively unformed. The majority of the tissue is mesenchymal cells.Early air spaces are being developed. On D5, air spaces are clearlyidentifiable, with thinning of the mesenchymal tissue and proliferationof the lung epithelium required to cover the enlarging air spaces.

[0357] Using HER2 staining at D0 and D5, it was established that HER 2is uniformly present on D0 lung tissue throughout the lung epithelialtissue. No expression was present in the mesenchymal tissue. By D5, HER2remains uniform and restricted to the pulmonary epithelium.

[0358] Using HER3 staining control at D0 and D5, HER3 was identifiablein D0 lung tissue. The staining was relatively less than HER2, and wasnot homogenous, suggesting that there are specific epithelial areasexpressing the HER3 receptor. By D5, expression had become morehomogenous throughout the lung epithelium, but clearly not uniform.Expression remained epithelium specific.

Example 5

[0359] Preparation of rHRGβ1₁₇₇₋₂₄₄

[0360] The EGF-like domain fragment HRG-β1 177-244 was amplified fromvector pHL89 (which is described in Holmes et al., Science 256:1205-1210(1992)) by PCR with primers having Nsil/XbaI-containing overhangs. Thefragment was inserted into phagemid display vector pam-g3 by restrictiondigest-ligation at the same sites to generate construct pHRG2-g3(177-244). pam-g3 was a derivative of phGHam-g3, which was designed forphage display of human growth hormone (hGH) and was described in Lowmanet al., Biochemistry 30:10832-10838 (1991). pam-g3 was produced byremoving the hGH gene present in phGHam-g3 and replacing this gene witha stuffer fragment, which provides space for cleavage at the restrictionsites used for cloning. The HRG-β1 fragment was attached to residue 247of pIII.

[0361] The HRG-β1 EGF-like domain expressed from the above-describedconstruct is designated by removing the “p” and the “−g3” that appear inthe name of the construct. Thus, the HRG-β1 EGF-like domain expressedfrom the pHRG2-g3 construct is designated “HRG2.”

[0362] The domain was displayed monovalently on phage as a pIII fusionprotein, as described by Bass et al., Proteins 8:309-314 (1990).

[0363] Similarly, variants HRG-β1₁₄₇₋₂₂₇, HRG-β1_(147-244,) andHRG-β1₁₇₇₋₂₂₇ were prepared and expressed as described above.

Example 6

[0364] rHRGβ1_(177-244A) Causes Accelerated Lung Development

[0365] Exogenous rHRGβ1₁₇₇₋₂₄₄ caused accelerated lung development invitro. To determine if the expressed HER2/HER3 receptors were functionaland the role of HRG stimulation during lung development in vitro,rHRGβ1₁₇₇₋₂₄₄ was added to the in vitro culture at 10 nM. Tissue washarvested at D5, snap frozen, and 5 micron sections were cut foranalysis.

[0366] The morphology, in comparison to the untreated control specimenswas grossly different. There was marked proliferation of the epithelium.Air spaces which are typically lined with a single cell layer now had a2-3 cell thickness. The changes were dose dependent with more epithelialcell response with higher concentrations. HER2 and HER3 were stillidentifiable in the epithelium only.

Example 7

[0367] Human Lung Differentiation

[0368] Differentiation of human lung epithelial cells occurs after HRGtreatment. Differentiation was measured by Surfactant Protein A (SPA)production. All sections were stained for SPA. Human lung explantstained for SPA with stain localizing in epithelial cells of theprealveolar ducts. Human lung explant exposed to 10 nM HRG showed aneffect on SPA production. As a differentiation control, a lung explantwas exposed to 1 mM dibutyryl cAMP. A negative control was also run.                175                 180 gag aag gag aaa act ttc tgt gtgaat gga ggg gag tgc 1083 Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly GluCys     185                 190                 195 ttc atg gtg aaa gacctt tca aac 000 tcg aga tac ttg 1122 Phe Met Val Lys Asp Leu Ser Asn ProSer Arg Tyr Leu             200                 205 tgc aag tgt cca aatgag ttt act ggt gat agc tgc caa 1161 Cys Lys Cys Pro Asn Glu Phe Thr GlyAsp Arg Cys Gln 210                 215                 220 aac tac gtaatg gcc agc ttc tac aag gcg gag gag ctg 1200 Asn Tyr Val Met Ala Ser PheTyr Lys Ala Glu Glu Leu        225                 230                 235 tac cag aag aga gtgctg acc ata acc ggc atc tgc atc 1239 Tyr Gln Lys Arg Val Leu Thr Ile ThrGly Ile Cys Ile                 240                 245 gcc ctc ctt gtggtc ggc atc atg tgt gtg gtg gcc tac 1278 Ala Leu Leu Val Val Gly Ile MetCys Val Val Ala Tyr     250                 255                 260 tgcaaa acc aag aaa cag cgg aaa aag ctg cat gac cgt 1317 Cys Lys Thr Lys LysGln Arg Lys Lys Leu His Asp Arg             265                 270 cttcgg cag agc ctt cgg tct gaa cga aac aat atg atg 1356 Leu Arg Gln Ser LeuArg Ser Glu Arg Asn Asn Met Met275                 280                 285 aac att gcc aat ggg cct caccat cct aac cca ccc ccc 1395 Asn Ile Ala Asn Gly Pro His His Pro Asn ProPro Pro         290                 295                 300 gag aat gtccag ctg gtg aat caa tac gta tct aaa aac 1434 Glu Asn Val Gln Leu Val AsnGln Tyr Val Ser Lys Asn                 305                 310 gtc atctcc agt gag cat att gtt gag aga gaa gca gag 1473 Val Ile Ser Ser Glu HisIle Val Glu Arg Glu Ala Glu    315                 320                 325 aca tcc ttt tcc acc agtcac tat act tcc aca gcc cat 1512 Thr Ser Phe Ser Thr Ser His Tyr Thr SerThr Ala His             330                 335 cac tcc act act gtc acccag act cct agc cac agc tgg 1551 His Ser Thr Thr Val Thr Gln Thr Pro SerHis Ser Trp 340                 345                 350 agc aac gga cacact gaa agc atc ctt tcc gaa agc cac 1590 Ser Asn Gly His Thr Glu Ser IleLeu Ser Glu Ser His         355                 360                 365tct gta atc gtg atg tca tcc gta gaa aac agt agg cac 1629 Ser Val Ile ValMet Ser Ser Val Glu Asn Ser Arg His                370                 375 agc agc cca act ggg ggc cca agagga cgt ctt aat ggc 1668 Ser Ser Pro Thr Gly Gly Pro Arg Gly Arg Leu AsnGly     380                 385                 390 aca gga ggc cct cgtgaa tgt aac agc ttc ctc agg cat 1707 Thr Gly Gly Pro Arg Glu Cys Asn SerPhe Leu Arg His             395                 400 gcc aga gaa acc cctgat tcc tac cga gac tct cct cat 1746 Ala Arg Glu Thr Pro Asp Ser Tyr ArgAsp Ser Pro His 405                 410                 415 agt gaa aggtaaaa ccgaaggcaa agctactgca gaggagaaac 1790 Ser Glu Arg         420tcagtcagag aatccctgtg agcacctgcg gtctcacctc aggaaatcta 1840 ctctaatcagaataaggggc ggcagttacc tgttctagga gtgctcctag 1890 ttgatgaagt catctctttgtttgacggaa cttatttctt ctgagcttct 1940 ctcgtcgtcc cagtgactga caggcaacagactcttaaag agctgggatg 1990 ctttgatgcg gaaggtgcag cacatggagt ttccagctctggccatgggc 2040 tcagacccac tcggggtctc agtgtcctca gttgtaacat tagagagatg2090 gcatcaatgc ttgataagga cccttctata attccaattg ccagttatcc 2140aaactctgat tcggtggtcg agctggcctc gtgttcttat ctgctaaccc 2190 tgtcttaccttccagcctca gttaagtcaa atcaagggct atgtcattgc 2240 tgaatgtcat ggggggcaactgcttgccct ccaccctata gtatctattt 2290 tatgaaattc caagaaggga tgaataaataaatctcttgg atgctgcgtc 2340 tggcagtctt cacgggtggt tttcaaagca gaaaaaaaaaaaaaaaaaaa 2390 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 2431 <210>11 <211> 768 <212> PRT <213> Homo sapiens <400> 11 Met Asp Val Lys GluArg Lys Pro Tyr Arg Ser Leu Thr Arg Arg  1               5                  10                  15 Arg Asp AlaGlu Arg Arg Tyr Thr Ser Ser Ser Ala Asp Ser Glu                 20                  25                  30 Glu Gly LysAla Pro Gln Lys Ser Tyr Ser Ser Ser Glu Thr Leu                 35                  40                  45 Lys Ala TyrAsp Gln Asp Ala Arg Leu Ala Tyr Gly Ser Arg Val                 50                  55                  60 Lys Asp IleVal Pro Gln Glu Ala Glu Glu Phe Cys Arg Thr Gly                 65                  70                  75 Ala Asn PheThr Leu Arg Glu Leu Gly Leu Glu Glu Val Thr Pro                 80                  85                  90 Pro His GlyThr Leu Tyr Arg Thr Asp Ile Gly Leu Pro His Cys                 95                 100                 105 Gly Tyr SerMet Gly Ala Gly Ser Asp Ala Asp Met Glu Ala Asp                110                 115                 120 Thr Val LeuSer Pro Glu His Pro Val Arg Leu Trp Gly Arg Ser                125                 130                 135 Thr Arg SerGly Arg Ser Ser Cys Leu Ser Ser Arg Ala Asn Ser                140                 145                 150 Asn Leu ThrLeu Thr Asp Thr Glu His Glu Asn Thr Glu Thr Asp                155                 160                 165 His Pro GlyGly Leu Gln Asn His Ala Arg Leu Arg Thr Pro Pro                170                 175                 180 Pro Pro LeuSer His Ala His Thr Pro Asn Gln His His Ala Ala                185                 190                 195 Ser Ile AsnSer Leu Asn Arg Gly Asn Phe Thr Pro Arg Ser Asn                200                 205                 210 Pro Ser ProAla Pro Thr Asp His Ser Leu Ser Gly Glu Pro Pro                215                 220                 225 Ala Gly GlyAla Gln Glu Pro Ala His Ala Gln Glu Asn Trp Leu                230                 235                 240 Leu Asn SerAsn Ile Pro Leu Glu Thr Arg Asn Leu Gly Lys Gln                245                 250                 255 Pro Phe LeuGly Thr Leu Gln Asp Asn Leu Ile Glu Met Asp Ile                260                 265                 270 Leu Gly AlaSer Arg His Asp Gly Ala Tyr Ser Asp Gly His Phe                275                 280                 285 Leu Phe LysPro Gly Gly Thr Ser Pro Leu Phe Cys Thr Thr Ser                290                 295                 300 Pro Gly TyrPro Leu Thr Ser Ser Thr Val Tyr Ser Pro Pro Pro                305                 310                 315 Arg Pro LeuPro Arg Ser Thr Phe Ala Arg Pro Ala Phe Asn Leu                320                 325                 330 Lys Lys ProSer Lys Tyr Cys Asn Trp Lys Cys Ala Ala Leu Ser                335                 340                 345 Ala Ile ValIle Ser Ala Thr Leu Val Ile Leu Leu Ala Tyr Phe                350                 355                 360 Val Ala MetHis Leu Phe Gly Leu Asn Trp His Leu Gln Pro Met                365                 370                 375 Glu Gly GlnMet Tyr Glu Ile Thr Glu Asp Thr Ala Ser Ser Trp                380                 385                 390 Pro Val ProThr Asp Val Ser Leu Tyr Pro Ser Gly Gly Thr Gly                395                 400                 405 Leu Glu ThrPro Asp Arg Lys Gly Lys Gly Thr Thr Glu Gly Lys                410                 415                 420 Pro Ser SerPhe Phe Pro Glu Asp Ser Phe Ile Asp Ser Gly Glu                425                 430                 435 Ile Asp ValGly Arg Arg Ala Ser Gin Lys Ile Pro Pro Gly Thr                440                 445                 450 Phe Trp ArgSer Gln Val Phe Ile Asp His Pro Val His Leu Lys                455                 460                 465 Phe Asn ValSer Leu Gly Lys Ala Ala Leu Val Gly Ile Tyr Gly                470                 475                 480 Arg Lys GlyLeu Pro Pro Ser His Thr Gln Phe Asp Phe Val Glu                485                 490                 495 Leu Leu AspGly Arg Arg Leu Leu Thr Gln Glu Ala Arg Ser Leu                500                 505                 510 Glu Gly ThrPro Arg Gln Ser Arg Gly Thr Val Pro Pro Ser Ser                515                 520                 525 His Glu ThrGly Phe Ile Gln Tyr Leu Asp Ser Gly Ile Trp His                530                 535                 540 Leu Ala PheTyr Asn Asp Gly Lys Glu Ser Glu Val Val Ser Phe                545                 550                 555 Leu Thr ThrAla Ile Ala Leu Pro Pro Arg Leu Lys Glu Met Lys                560                 565                 570 Ser Gln GluSer Ala Ala Gly Ser Lys Leu Val Leu Arg Cys Glu                575                 580                 585 Thr Ser SerGlu Tyr Ser Ser Leu Arg Phe Lys Trp Phe Lys Asn                590                 595                 600 Gly Asn GluLeu Asn Arg Lys Asn Lys Pro Gln Asn Ile Lys Ile                605                 610                 615 Gln Lys LysPro Gly Lys Ser Glu Leu Arg Ile Asn Lys Ala Ser                620                 625                 630 Leu Ala AspSer Gly Glu Tyr Met Cys Lys Val Ile Ser Lys Leu                635                 640                 645 Gly Asn AspSer Ala Ser Ala Asn Ile Thr Ile Val Glu Ser Asn                650                 655                 660 Glu Ile IleThr Gly Met Pro Ala Ser Thr Glu Gly Ala Tyr Val                665                 670                 675 Ser Ser GluSer Pro Ile Arg Ile Ser Val Ser Thr Glu Gly Ala                680                 685                 690 Asn Thr SerSer Ser Thr Ser Thr Ser Thr Thr Gly Thr Ser His                695                 700                 705 Leu Val LysCys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly                710                 715                 720 Gly Glu CysPhe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr                725                 730                 735 Leu Cys LysCys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn                740                 745                 750 Tyr Val MetAla Ser Phe Tyr Ser Thr Ser Thr Pro Phe Leu Ser                755                 760                 765 Leu Pro Glu        768 <210> 12 <211> 3111 <212> DNA <213> Homo sapiens <400> 12gggtaccatg ggtcggtgag cgcgtttccc gcctgagcgc aactagcggc 50 gggtcgtgggcacctccaga aaagatcccg caccatcctc caggatccaa 100 tggccttgga gagagggctgcagggcccac ggacattgct gactcttcag 150 aacgtgctga catggagcca ggtagactgaaattatcatg tgtccaaatt 200 aaaattgcat acttcaagga ttatttgaag gactattcttagaccctttt 250 aagaagattt aaagaaaaac cactcggccc tgagtgcggc gaggaccctg300 tttgtggatg tggaggagcg cgggccggag gco    atg gac gtg 342                                        Met Asp Val                                          1 aag gag agg aag cct tac cgctcg ctg acc cgg cgc cgc 381 Lys Glu Arg Lys Pro Tyr Arg Ser Leu Thr ArgArg Arg       5                  10                  15 gac gcc gag cgccgc tac acc agc tcg tcc gcg gac agc 420 Asp Ala Glu Arg Arg Tyr Thr SerSer Ser Ala Asp Ser              20                  25 gag gag ggc aaagcc ccg cag aaa tcg tac agc tcc agc 459 Glu Glu Gly Lys Ala Pro Gln LysSer Tyr Ser Ser Ser  30                  35                  40 gag accctg aag gcc tac gac cag gac gcc cgc cta gcc 498 Glu Thr Leu Lys Ala TyrAsp Gln Asp Ala Arg Leu Ala         45                  50                  55 tat ggc agc cgc gtcaag gac att gtg ccg cag gag gcc 537 Tyr Gly Ser Arg Val Lys Asp Ile ValPro Gln Glu Ala                  60                  65 gag gaa ttc tgccgc aca ggt gcc aac ttc acc ctg cgg 576 Glu Glu Phe Cys Arg Thr Gly AlaAsn Phe Thr Leu Arg      70                  75                  80 gagctg ggg ctg gaa gaa gta acg ccc cct cac ggg acc 615 Glu Leu Gly Leu GluGlu Val Thr Pro Pro His Gly Thr              85                  90 ctgtac cgg aca gac att ggc ctc ccc cac tgc ggc tac 654 Leu Tyr Arg Thr AspIle Gly Leu Pro His Cys Gly Tyr 95                 100                 105 tcc atg ggg gct ggc tct gatgcc gac atg gag gct gac 693 Ser Met Gly Ala Gly Ser Asp Ala Asp Met GluAla Asp         110                 115                 120 acg gtg ctgtcc cct gag cac ccc gtg cgt ctg tgg ggc 732 Thr Val Leu Ser Pro Glu HisPro Val Arg Leu Trp Gly                 125                 130 cgg agcaca cgg tca ggg cgc agc tcc tgc ctg tcc agc 771 Arg Ser Thr Arg Ser GlyArg Ser Ser Cys Leu Ser Ser    135                 140                 145 cgg gcc aat tcc aat ctcaca ctc acc gac acc gag cat 810 Arg Ala Asn Ser Asn Leu Thr Leu Thr AspThr Glu His             150                 155 gaa aac act gag act gatcat ccg ggc ggc ctg cag aac 849 Glu Asn Thr Glu Thr Asp His Pro Gly GlyLeu Gln Asn 160                 165                 170 cac gcg cgg ctccgg acg ccg ccg ccg ccg ctc tcg cac 888 His Ala Arg Leu Arg Thr Pro ProPro Pro Leu Ser His         175                 180                 185gcc cac acc ccc aac cag cac cac gcg gcc tcc att aac 927 Ala His Thr ProAsn Gln His His Ala Ala Ser Ile Asn                190                 195 tcc ctg aac cgg ggc aac ttc acgccg agg agc aac ccc 966 Ser Leu Asn Arg Gly Asn Phe Thr Pro Arg Ser AsnPro     200                 205                 210 agc ccg gcc ccc acggac cac tcg ctc tcc gga gag ccc 1005 Ser Pro Ala Pro Thr Asp His Ser LeuSer Gly Glu Pro             215                 220 cct gcc ggc ggc gcccag gag cct gcc cac gcc cag gag 1044 Pro Ala Gly Gly Ala Gln Glu Pro AlaHis Ala Gln Glu 225                 230                 235 aac tgg ctgctc aac agc aac atc ccc ctg gag acc aga 1083 Asn Trp Leu Leu Asn Ser AsnIle Pro Leu Glu Thr Arg        240                 245                 250 aac cta ggc aag cagcca ttc cta ggg aca ttg cag gac 1122 Asn Leu Gly Lys Gln Pro Phe Leu GlyThr Leu Gln Asp                 255                 260 aac ctc att gagatg gac att ctc ggc gcc tcc cgc cat 1161 Asn Leu Ile Glu Met Asp Ile LeuGly Ala Ser Arg His     265                 270                 275 gatggg gct tac agt gac ggg cac ttc ctc ttc aag cct 1200 Asp Gly Ala Tyr SerAsp Gly His Phe Leu Phe Lys Pro             280                 285 ggaggc acc tcc ccg ctc ttc tgc acc aca tca cca ggg 1239 Gly Gly Thr Ser ProLeu Phe Cys Thr Thr Ser Pro Gly290                 295                 300 tac cca ctg acg tcc agc acagtg tac tct cct ccg ccc 1278 Tyr Pro Leu Thr Ser Ser Thr Val Tyr Ser ProPro Pro         305                 310                 315 cga ccc ctgccc cgc agc acc ttc gcc cgg ccg gcc ttt 1317 Arg Pro Leu Pro Arg Ser ThrPhe Ala Arg Pro Ala Phe                 320                 325 aac ctcaag aag ccc tcc aag tac tgt aac tgg aag tgc 1356 Asn Leu Lys Lys Pro SerLys Tyr Cys Asn Trp Lys Cys    330                 335                 340 gca gcc ctg agc gcc atcgtc atc tca gcc act ctg gtc 1395 Ala Ala Leu Ser Ala Ile Val Ile Ser AlaThr Leu Val             345                 350 atc ctg ctg gca tac tttgtg gcc atg cac ctg ttt ggc 1434 Ile Leu Leu Ala Tyr Phe Val Ala Met HisLeu Phe Gly 355                 360                 365 cta aac tgg cacctg cag ccg atg gag ggg cag atg tat 1473 Leu Asn Trp His Leu Gln Pro MetGlu Gly Gln Met Tyr         370                 375                 380gag atc acg gag gac aca gcc agc agt tgg cct gtg cca 1512 Glu Ile Thr GluAsp Thr Ala Ser Ser Trp Pro Val Pro                385                 390 acc gac gtc tcc cta tac ccc tcaggg ggc act ggc tta 1551 Thr Asp Val Ser Leu Tyr Pro Ser Gly Gly Thr GlyLeu     395                 400                 405 gag acc cct gac aggaaa ggc aaa gga acc aca gaa gga 1590 Glu Thr Pro Asp Arg Lys Gly Lys GlyThr Thr Glu Gly             410                 415 aag ccc agt agt ttcttt cca gag gac agt ttc ata gat 1629 Lys Pro Ser Ser Phe Phe Pro Glu AspSer Phe Ile Asp 420                 425                 430 tct gga gaaatt gat gtg gga agg cga gct tcc cag aag 1668 Ser Gly Glu Ile Asp Val GlyArg Arg Ala Ser Gln Lys        435                 440                 445 att cct cct ggc actttc tgg aga tct caa gtg ttc ata 1707 Ile Pro Pro Gly Thr Phe Trp Arg SerGln Val Phe Ile                 450                 455 gac cat cct gtgcat ctg aaa ttc aat gtg tct ctg gga 1746 Asp His Pro Val His Leu Lys PheAsn Val Ser Leu Gly     460                 465                 470 aaggca gcc ctg gtt ggc att tat ggc aga aaa ggc ctc 1785 Lys Ala Ala Leu ValGly Ile Tyr Gly Arg Lys Gly Leu             475                 480 cctcct tca cat aca cag ttt gac ttt gtg gag ctg ctg 1824 Pro Pro Ser His ThrGln Phe Asp Phe Val Glu Leu Leu485                 490                 495 gat ggc agg agg ctc cta acccag gag gcg cgg agc cta 1863 Asp Gly Arg Arg Leu Leu Thr Gln Glu Ala ArgSer Leu         500                 505                 510 gag ggg accccg cgc cag tct cgg gga act gtg ccc ccc 1902 Glu Gly Thr Pro Arg Gln SerArg Gly Thr Val Pro Pro                 515                 520 tcc agccat gag aca ggc ttc atc cag tat ttg gat tca 1941 Ser Ser His Glu Thr GlyPhe Ile Gln Tyr Leu Asp Ser    525                 530                 535 gga atc tgg cac ttg gctttt tac aat gac gga aag gag 1980 Gly Ile Trp His Leu Ala Phe Tyr Asn AspGly Lys Glu             540                 545 tca gaa gtg gtt tcc tttctc acc act gcc att gcc ttg 2019 Ser Glu Val Val Ser Phe Leu Thr Thr AlaIle Ala Leu 550                 555                 560 cct ccc cga ttgaaa gag atg aaa agc cag gaa tcg gct 2058 Pro Pro Arg Leu Lys Glu Met LysSer Gln Glu Ser Ala         565                 570                 575gca ggt tcc aaa cta gtc ctt cgg tgt gaa aco agt tct 2097 Ala Gly Ser LysLeu Val Leu Arg Cys Glu Thr Ser Ser                580                 585 gaa tac tcc tct ctc aga ttc aagtgg ttc aag aat ggg 2136 Glu Tyr Ser Ser Leu Arg Phe Lys Trp Phe Lys AsnGly     590                 595                 600 aat gaa ttg aat cgaaaa aac aaa cca caa aat atc aag 2175 Asn Glu Leu Asn Arg Lys Asn Lys ProGln Asn Ile Lys             605                 610 ata caa aaa aag ccaggg aag tca gaa ctt cgc att aac 2214 Ile Gln Lys Lys Pro Gly Lys Ser GluLeu Arg Ile Asn 615                 620                 625 aaa gca tcactg gct gat tct gga gag tat atg tgc aaa 2253 Lys Ala Ser Leu Ala Asp SerGly Glu Tyr Met Cys Lys        630                 635                 640 gtg atc agc aaa ttagga aat gac agt gcc tct gac aat 2292 Val Ile Ser Lys Leu Gly Asn Asp SerAla Ser Ala Asn                 645                 650 atc acc atc gtggaa tca aac gag atc atc act ggt atg 2331 Ile Thr Ile Val Glu Ser Asn GluIle Ile Thr Gly Met     655                 660                 665 ccagcc tca act gaa gga gca tat gtg tct tca gag tct 2370 Pro Ala Ser Thr GluGly Ala Tyr Val Ser Ser Glu Ser             670                 675 cccatt aga ata tca gta tcc aca gaa gga gca aat act 2409 Pro Ile Arg Ile SerVal Ser Thr Glu Gly Ala Asn Thr680                 685                 690 tct tca tct aca tct aca tccacc act ggg aca agc cat 2448 Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly ThrSer His         695                 700                 705 ctt gta aaatgt gcg gag aag gag aaa act ttc tgt gtg 2487 Leu Val Lys Cys Ala Glu LysGlu Lys Thr Phe Cys Val                 710                 715 aat ggaggg gag tgc ttc atg gtg aaa gac ctt tca aac 2526 Asn Gly Gly Glu Cys PheMet Val Lys Asp Leu Ser Asn    720                 725                 730 ccc tcg aga tac ttg tgcaag tgc cca aat gag ttt act 2565 Pro Ser Arg Tyr Leu Cys Lys Cys Pro AsnGlu Phe Thr             735                 740 ggt gat cgc tgc caa aactac gta atg gcc agc ttc tac 2604 Gly Asp Arg Cys Gln Asn Tyr Val Met AlaSer Phe Tyr 745                 750                 755 agt acg tcc actccc ttt ctg tct ctg cct gaa tag 2640 Ser Thr Ser Thr Pro Phe Leu Ser LeuPro Glu         760                 765         768 gagcatgctcagttggtgct gctttcttgt tgctgcatct cccctcagat 2690 tccacctaga gctagatgtgtcttaccaga tctaatattg actgcctctg 2740 cctgtcgcat gagaacatta acaaaagcaattgtattact tcctctgttc 2790 gcgactagtt ggctctgaga tactaatagg tgtgtgaggctccggatgtt 2840 tctggaattg atattgaatg atgtgataca aattgatagt caatatcaag2890 cagtgaaata tgataataaa ggcatttcaa agtctcactt ttattgataa 2940aataaaaatc attctactga acagtccatc ttctttatac aatgaccaca 2990 tcctgaaaagggtgttgcta agctgtaacc gatatgcact tgaaatgatg 3040 gtaagttaat tttgattcagaatgtgttat ttgtcacaaa taaacataat 3090 aaaaggaaaa aaaaaaaaaa a 3111 <210>13 <211> 1872 <212> DNA <213> Homo sapiens <400> 13 gaattcgggacagcctctcc tgccgccgct gctgctgccg ccgccgccac 50 cgccggctgg tcctccttctgcttttactt ctcctgcatg acagttgttt 100 tcttcatctg agcagacacc agcttcagatgctcgaggtg agaaacatgc 150 ctttcagttt gggctactgg tttacttaat taatcagccggcagctccgt 200 cgatctattt tcgtccctgt cctcttgacg agcccgggat ggtttggagt250 agcatttaaa agaactagaa aagtggccca gaaacagcag cttaaagaat 300tattacgata tactttgatt ttgtagttgc taggagcttt tcttcccccc 350 ttgcatctttctgaactctt cttgatttta ataatggcct tggacttgga 400 cgatttatcg atttccccctgtaagatgct gtatcatttg gttggggggg 450 cctctgcgtg gtaatggacc gtgagagcggccaggccttc ttctggaggt 500 gagccg atg gag att tat tcc cca gac atg tct gaggtc 539         Met Glu Ile Tyr Ser Pro Asp Met Ser Glu Val          1               5                  10 gcc gcc gag agg tcc tccagc ccc tcc act cag ctg agt 578 Ala Ala Glu Arg Ser Ser Ser Pro Ser ThrGln Leu Ser              15                  20 gca gac cca tct ctt gatggg ctt ccg gca gca gaa gac 617 Ala Asp Pro Ser Leu Asp Gly Leu Pro AlaAla Glu Asp  25                  30                  35 atg cca gag ccccag act gaa gat ggg aga acc cct gga 656 Met Pro Glu Pro Gln Thr Glu AspGly Arg Thr Pro Gly          40                  45                  50ctc gtg ggc ctg gcc gtg ccc tgc tgt gcg tgc cta gaa 695 Leu Val Gly LeuAla Val Pro Cys Cys Ala Cys Leu Glu                 55                  60 gct gag cgc ctg aga ggt tgc ctcaac tca gag aaa atc 734 Ala Glu Arg Leu Arg Gly Cys Leu Asn Ser Glu LysIle      65                  70                  75 tgc att gtc ccc atcctg gct tgc ctg gtc agc ctc tgc 773 Cys Ile Val Pro Ile Leu Ala Cys LeuVal Ser Leu Cys              80                  85 ctc tgc atc gcc ggcctc aag tgg gta ttt gtg gac aag 812 Leu Cys Ile Ala Gly Leu Lys Trp ValPhe Val Asp Lys  90                  95                 100 atc ttt gaatat gac tct cct act cac ctt gac cct ggg 851 Ile Phe Glu Tyr Asp Ser ProThr His Leu Asp Pro Gly        105                 110                 115 ggg tta ggc cag gaccct att att tct ctg gac gca act 890 Gly Leu Gly Gln Asp Pro Ile Ile SerLeu Asp Ala Thr                 120                 125 gct gcc tca gctgtg tgg gtg tcg tct gag gca tac act 929 Ala Ala Ser Ala Val Trp Val SerSer Glu Ala Tyr Thr     130                 135                 140 tcacct gtc tct agg gct caa tct gaa agt gag gtt caa 968 Ser Pro Val Ser ArgAla Gln Ser Glu Ser Glu Val Gln             145                 150 gttaca gtg caa ggt gac aag gct gtt gtc tcc ttt gaa 1007 Val Thr Val Gln GlyAsp Lys Ala Val Val Ser Phe Glu155                 160                 165 cca tca gcg gca ccg aca ccgaag aat cgt att ttt gcc 1046 Pro Ser Ala Ala Pro Thr Pro Lys Asn Arg IlePhe Ala         170                 175                 180 ttt tct ttcttg ccg tcc act gcg cca tcc ttc cct tca 1085 Phe Ser Phe Leu Pro Ser ThrAla Pro Ser Phe Pro Ser                 185                 190 ccc acccgg aac cct gag gtg aga acg ccc aag tca gca 1124 Pro Thr Arg Asn Pro GluVal Arg Thr Pro Lys Ser Ala    195                 200                 205 act cag cca caa aca acagaa act aat ctc caa act gct 1163 Thr Gln Pro Gln Thr Thr Glu Thr Asn LeuGln Thr Ala             210                 215 cct aaa ctt tct aca tctaca tcc acc act ggg aca agc 1202 Pro Lys Leu Ser Thr Ser Thr Ser Thr ThrGly Thr Ser 220                 225                 230 cat ctt gta aaatgt gcg gag aag gag aaa act ttc tgt 1241 His Leu Val Lys cys Ala Glu LysGlu Lys Thr Phe Cys         235                 240                 245gtg aat gga ggg gag tgc ttc atg gtg aaa gac ctt tca 1280 Val Asn Gly GlyGlu cys Phe Met Val Lys Asp Leu Ser                250                 255 aac ccc tag aga tac ttg tgc aagtgc cca aat gag ttt 1319 Asn Pro Ser Arg Tyr Leu Cys Lys Cys Pro Asn GluPhe     260                 265                 270 act ggt gat agc tgccaa aac tac gta atg gac agc tta 1358 Thr Gly Asp Arg Cys Gln Asn Tyr ValMet Ala Ser Phe             275                 280 tac agt acg tcc actccc ttt atg tct ctg cct gaa taggag 1400 Tyr Ser Thr Ser Thr Pro Phe LeuSer Leu Pro Glu 285                 290                 295 296catgctcagt tggtgatgct ttattgttgc tgcatatccc atcagattcc 1450 acatagagctagatgtgtct taccagatat aatattgaat gcctctgcct 1500 gtcgaatgag aaaattaaaaaaagcaattg tattaattcc tctgttcgcg 1550 aatagttggc tctgagatac taataggtgtgtgaggctcc ggatgtttct 1600 ggaattgata ttgaatgatg tgatacaaat tgatagtcaatatcaagcag 1650 tgaaatatga taataaaggc atttaaaagt ctcactttta ttgataaaat1700 aaaaatcatt ctactgaaca gtccatcttc tttatacaat gaccacatcc 1750tgaaaagggt gttgctaagc tgtaaccgat atgcacttga aatgatggta 1800 agttaattttgattcagaat gtgttatttg tcacaaataa acataataaa 1850 aggaaaaaaa aaacaagaattc 1872 <210> 14 <211> 296 <212> PRT <213> Homo sapiens <400> 14 Met GluIle Tyr Ser Pro Asp Met Ser Glu Val Ala Ala Glu Arg  1               5                  10                  15 Ser Ser SerPro Ser Thr Gln Leu Ser Ala Asp Pro Ser Leu Asp                 20                  25                  30 Gly Leu ProAla Ala Glu Asp Met Pro Glu Pro Gln Thr Glu Asp                 35                  40                  45 Gly Arg ThrPro Gly Leu Val Gly Leu Ala Val Pro Cys Cys Ala                 50                  55                  60 Cys Leu GluAla Glu Arg Leu Arg Gly Cys Leu Asn Ser Glu Lys                 65                  70                  75 Ile Cys IleVal Pro Ile Leu Ala Cys Leu Val Ser Leu Cys Leu                 80                  85                  90 Cys Ile AlaGly Leu Lys Trp Val Phe Val Asp Lys Ile Phe Glu                 95                 100                 105 Tyr Asp SerPro Thr His Leu Asp Pro Gly Gly Leu Gly Gln Asp                110                 115                 120 Pro Ile IleSer Leu Asp Ala Thr Ala Ala Ser Ala Val Trp Val                125                 130                 135 Ser Ser GluAla Tyr Thr Ser Pro Val Ser Arg Ala Gln Ser Glu                140                 145                 150 Ser Glu ValGin Val Thr Val Gln Gly Asp Lys Ala Val Val Ser                155                 160                 165 Phe Glu ProSer Ala Ala Pro Thr Pro Lys Asn Arg Ile Phe Ala                170                 175                 180 Phe Ser PheLeu Pro Ser Thr Ala Pro Ser Phe Pro Ser Pro Thr                185                 190                 195 Arg Asn ProGlu Val Arg Thr Pro Lys Ser Ala Thr Gln Pro Gln                200                 205                 210 Thr Thr GluThr Asn Leu Gln Thr Ala Pro Lys Leu Ser Thr Ser                215                 220                 225 Thr Ser ThrThr Gly Thr Ser His Leu Val Lys Cys Ala Glu Lys                230                 235                 240 Glu Lys ThrPhe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys                245                 250                 255 Asp Leu SerAsn Pro Ser Arg Tyr Leu Cys Lys Cys Pro Asn Glu                260                 265                 270 Phe Thr GlyAsp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr                275                 280                 285 Ser Thr SerThr Pro Phe Leu Ser Leu Pro Glu                 290                 295296

We claim:
 1. A method of inducing epithelial cell growth and/orproliferation, comprising contacting a normal epithelial cell whichexpresses HER2, HER3 and/or HER4 receptors with an effective amount ofan isolated ligand which activates HER2, HER3, HER4 receptors or acombination thereof.
 2. The method of claim 1 , wherein the activatingligand is a heregulin (HRG) polypeptide, HRG variant, HRG agonistantibody or fragment thereof capable of binding to the HER2, HER3 and/orHER4 receptor.
 3. The method of claim 2 , wherein the activating ligandis human HRG or a fragment thereof.
 4. The method of claim 2 , whereinthe activating ligand is selected from the group consisting of HRG-α,-β1, -β2, -β2-like, and -β3 and fragments thereof.
 5. The method ofclaim 2 , wherein the activating ligand is γ-HRG or a fragment thereof.6. The method of claim 2 , wherein the activating ligand is recombinanthuman HRG or a fragment thereof.
 7. The method of claim 2 , wherein theactivating ligand is sensory and motor neuron derived factor (SMDF) or afragment thereof.
 8. The method of claim 1 , wherein the activatingligand is administered at a daily dose of about 1 μg/kg to 100 mg/kg. 9.The method of claim 2 , wherein the activating ligand is an agonistantibody.
 10. The method of claim 1 , wherein the contacting is byadministration to a patient in need thereof.
 11. The method of claim 6 ,wherein the HRG is rHRG-β1-177-244.
 12. The method of claim 1 , whereinthe epithelial cell is a lung cell.
 13. The method of claim 1 whereinthe epithelial cell expresses HER2/HER3, HER2/HER4, HER3/HER4, HER3 orHER4.
 14. A method of increasing lung surfactant protein A, comprisingadministering to a patient in need thereof an effective amount of anisolated HER2, HER3 and/or HER4 activating ligand.
 15. The method ofclaim 14 , wherein the activating ligand is a heregulin (HRG)polypeptide, HRG variant, HRG agonist antibody or fragment thereofcapable of binding to the HER2, HER3 and/or HER4 receptor.
 16. A methodof treating chronic obstructive pulmonary disease, comprisingadministering to a patient in need thereof an effective amount of anisolated HER2, HER3 and/or HER4 activating ligand.
 17. The method ofclaim 16 , wherein the activating ligand is a heregulin (HRG)polypeptide, HRG variant, HRG agonist antibody or fragment thereofcapable of binding to the HER2, HER3 and/or HER4 receptor.
 18. A methodof treating respiratory distress or emphysema, comprising administeringto a patient in need thereof an effective amount of an isolated HER2,HER3 and/or HER4 activating ligand.
 19. The method of claim 18 , whereinthe activating ligand is a heregulin (HRG) polypeptide, HRG variant, HRGagonist antibody or fragment thereof capable of binding to the HER2,HER3 and/or HER4 receptor.
 20. A method, comprising the steps of: (a)obtaining a normal epithelial cell sample from a mammal; (b) contactingthe sample with a ligand which activates HER2, HER3, HER4 or acombination thereof to induce growth and/or proliferation of epithelialcells in the sample and to obtain an expanded sample; and (c)re-introducing the expanded sample into the mammal.